Pickering Emulsion Stabilized by Immobilized Bienzyme Nanoparticles

Article pubs.acs.org/IECR

Pickering Emulsion Stabilized by Immobilized Bienzyme Nanoparticles: A Novel and Robust System for Enzymatic Purification of Isomaltooligosaccharide Jing Gao, Dan Li, Yanjun Jiang,* Li Ma, Liya Zhou, and Ying He School of Chemical Engineering and Technology, Hebei University of Technology, Tianjin 300130, P.R. China S Supporting Information *

ABSTRACT: A bienzyme system aiming at removal of glucose from isomaltooligosaccharide (IMO) was developed in this study. In this bienzymatic system, the first enzyme (glucose oxidase, GOD) catalyzed glucose to generate H2O2 and gluconic acid, while the second one (catalase, CAT) broke down the undesired H2O2 to water and oxygen, which can reduce the peroxideinduced degradation of GOD. For improving the catalytic activity, realizing the recyclability, and simplifying the separation operations of the enzymes, a Pickering emulsion stabilized by GOD- and CAT-containing silica particles was constructed. The optimal reaction temperature and pH were identified as 45 °C and 6.0, respectively. Under such conditions, the removal efficiency of glucose could be maintained at ca. 73.57%, even after five cycles in repeated batch operations. It is believed that the proposed process of glucose removal with biocatalytic Pickering emulsion is promising for the purification of IMO on an industrial scale. and used in some food-processing fields.14−16 It is well-known that the presence of CAT can reduce the inactivation of GOD, since the product of the GOD-catalyzed reaction (H2O2) is the substrate of CAT. However, until now, there has not been a report about the removal of glucose from IMO using this GOD−CAT system as catalyst. Compared to the single enzymatic process, some advantage may be obtained when this GOD−CAT system was used in removal of glucose from IMO: (1) the first enzyme (GOD) generates H2O2 by catalyzing glucose, while the second one (CAT; hydrogen peroxide:hydrogen peroxide oxidoreductase, EC 1.11.1.6, whose active center, composed of a heme with a tyrosine as the fifth ligand to the iron, a histidine, and an asparagine, is deeply buried in a highly conserved β-barrel core structure17) breaks down the undesired H2O2 to water and oxygen,14−16 and the regenerated oxygen can be used to assist the glucose oxidation catalyzed by GOD; (2) the removal of H2O2 can reduce the peroxide-induced degradation of GOD; and (3) gluconic acid, the byproduct, which can be separated easily by adsorption or precipitation, can also find applications in pharmaceutical and food industries as an acidity regulator and in mineral supplements.18 The concomitant production of gluconic acid could significantly enhance the commercial viability of this proposed process for the removal of glucose on an industrial scale.19 Recently, immobilization of enzymes on nanoparticles as nanobiocatalysts has received increasing attention for in vitro biotransformation because of the higher enzyme loading and higher catalytic performance.20,21 However, the facile recycling of these nanobiocatalysts remains a significant challenge in

1. INTRODUCTION Isomaltooligosaccharides (IMO), with the advantages of possessing low sweetness, few calories, and other properties beneficial to health, have been widely used in various foods and drinks.1−4 However, the presence of simple sugars such as glucose may seriously affect the function and commercial value of IMO. Removal of glucose provides IMO with several advantages, such as increased viscosity, reduced sweetness and hygroscopicity, and fewer Maillard reactions during heat processing.5 Additionally, the absence of simple sugars can lower the sweetness and calorific value and allow the IMO to be included in diabetic foods.5 Generally, chromatographic processes are employed to remove simple sugars from IMO.1,6 These processes may not be cost-effective on an industrial scale, and the purified products still retain 5−10% of simple sugars.6,7 Further purification of IMO cannot be easily achieved with conventional operations such as extraction, adsorption, or precipitation. Although an immobilized cells method has been developed,5 to further increase the purity of IMO, it is necessary to develop a simple and efficient process for the removal of glucose. Theoretically, glucose in IMO can be removed enzymatically with glucose oxidase (GOD; β-D-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4),6 which can catalyze the oxidation of glucose to H2O2 and gluconic acid.8,9 GOD is a dimeric protein containing one tightly but noncovalently bound flavin adenine dinucleotide (FAD) per monomer as cofactor, and the key conserved active-site residues of GOD from Aspergillus niger are Glu-412, His-559, and His-516.10 However, GOD may be inactivated by H2O2, the concentration of which increases during the catalytic turnover.8,11 The inactivation mechanisms involve modification of certain methionine residues located at or near the active site12 and the attack of peroxide on the glucose−GOD complex.13 During the past few years, a system of glucose oxidase plus catalase (GOD−CAT) was constructed © 2014 American Chemical Society

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practical application because of their nanoscale sizes.20 As far as we know, centrifugation is the most commonly used strategy for recovering nanoparticles.22,23 However, the high energy consumption and low recovery efficiency often limited their applications. A Pickering emulsion, a kind of emulsion that is stabilized by colloidal nanoparticles, has attracted a surge of interest.24 When a Pickering emulsion is stabilized by enzymecontaining nanoparticles, this Pickering emulsion can be used as a kind of “biocatalyst system” without dramatically changing the nanobiocatalyst’s catalytic properties.25 Compared to the nanobiocatalysts, the recycling of the Pickering emulsion was easily. Previous reports confirmed that the creation of a Pickering emulsion stabilized by enzyme-containing silica particles could simplify the separation operation and, more importantly, enable the enzymes to be reusable and robust biocatalysts, which affords a promising approach to construct robust biocatalytic system.24−26 Thus, in the present study, Pickering emulsion stabilized by immobilized GOD and CAT nanoparticles was constructed and used to scavenge the glucose exists in IMO. Up to now, this is the first report concerning the preparation and application of the Pickering emulsion that stabilized by immobilized GOD and CAT nanoparticles. The effects of the parameters (activity ratio of GOD to CAT, the amount of Pickering emulsion, temperature, and pH) on the removal efficiency of glucose were studied, and the reusability of the Pickering emulsion was also investigated.

20 mL of toluene, and then the mixture was under ultrasonic processing to fully scatter the nanoparticles. Toluene (80 mL) and APTES (10 mL) were then added, and the above mixture was refluxed at 120 °C for 4 h. Amino-functionalized SiO2 particles were then obtained after washing with ethanol three times and drying under vacuum at room temperature for 12 h. To activate amino-functionalized SiO2 particles, 0.5 mL of glutaraldehyde solution was added into 0.5 mL of 10 mg/mL amino-functionalized SiO2 particles [dissolved in phosphate buffer solution (PBS, pH 7.0)], and the reaction was allowed to continue for different time periods. The glutaraldehydeactivated SiO2 particles were obtained after the mixture was centrifuged and cleaned up with PBS (pH 7.0) several times. For the immobilization of GOD and CAT, 0.5 mL of enzyme solution (dissolved in PBS, pH 5.5 for GOD and 7.0 for CAT) was added into the above-activated SiO2 and the reaction was allowed to continue for different time periods. The immobilized enzymes (GOD−SiO2 and CAT−SiO2) were obtained after centrifugation and washed with PBS several times. Effects of the concentration of glutaraldehyde, carrier activation time, enzyme concentration, and immobilization time on the enzyme activity were investigated. The stability and kinetic parameters of the enzymes were evaluated. 2.3. Preparation of a Pickering Emulsion Stabilized by Immobilized GOD and CAT Nanoparticles. A certain amount of GOD−SiO2 and CAT−SiO2 was dispersed in 20 mL of deionized water, 4 mL of plant oil was added to the dispersion, and the mixture was emulsified after homogenizing for 5 min, and then the Pickering emulsion stabilized by GOD−SiO2 and CAT−SiO2 particles was obtained. To obtain a stable Pickering emulsion, the weight ratio of the immobilized enzyme nanoparticles to oil was optimized and set as 1:230 (w/ w, 1 mg of nanoparticles/230 mg of oil). 2.4. Removal of Glucose Using the Biocatalytic Pickering Emulsion. A certain amount of the as-prepared Pickering emulsion was added to 5 mL of the IMO solution (500 mg/mL) and incubated at different temperatures for a certain time. The generated gluconic acid was removed through the addition of Ca(OH)2. High-performance liquid chromatography (HPLC) was used to detect the glucose that remained in the IMO after centrifugation and membrane filtration treatment of the reaction mixture. Samples (15 μL) were injected into an HPLC system (Agilent 1260, America) equipped with a column (4.6 × 250 mmol/L, Zorbax carbohydrate). The column was maintained at 30 °C and was eluted isocratically with a mobile phase consisting of acetonitrile and Milli-Q water at a volume ratio of 70:30 at a flow rate of 1 mL/min. The eluent was monitored with a refractive index (RI) detector.

2. MATERIALS AND METHODS 2.1. Materials. Nanosilica particles (fumed silica with diameter of 30 ± 5 nm, density of ca. 2.65 g/cm3, and purity ≥99.5%) were obtained from Shanghai Maikun Chemical Co., Ltd. (Shanghai, China). Glucose oxidase (GOD; EC 1.1.3.4, ≥100 U/mg) was obtained from Shanghai Sanjie Biological Technology Co., Ltd. (Shanghai, China). Catalase (CAT; EC 1.11.1.6, ≥2000−5000 U/mg) and horseradish peroxidase (HRP; EC 1.11.1.7, ≥150 U/mg) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Isomaltooligosaccharide (IMO; food grade, glucose content of 5.0%) was purchased from Shandong Baolingbao Co., Ltd. (Shangdong, China). Acetonitrile (chromatographically pure) was purchased from Tianjin Concord Science and Technology Co., Ltd. (Tianjin, China). 3-Aminopropyltriethoxysilane (APTES) was purchased from Beijing Shenda Fine Chemical Co., Ltd. (Beijing, China). All other reagents were of analytical grade and used without further purification. 2.2. Preparation of Immobilized GOD and CAT. The immobilization procedures, including the amino functionalization of the SiO2 nanoparticles with APTES, the activation of amino-functionalized SiO2 nanoparticles with glutaraldehyde, and the immobilization of GOD and CAT, are presented in Scheme 1. Typically, 1 g of the SiO2 nanoparticles was added to

3. RESULTS AND DISCUSSION 3.1. Characterization of the Immobilized Enzyme Nanoparticles and Pickering Emulsion. Silica nanoparticles were employed as carriers for immobilization of GOD and CAT. The FT-IR spectra of the silica, APTES-modified silica, and GOD−SiO2 and CAT−SiO2 nanoparticles were investigated (Figure 1). After the reaction with APTES, bands at 2927 and 1558 cm−1 were assigned to −CH2 stretching27−29 and −NH2 bending,27,30 respectively, which could confirm that APTES had been modified on the silica nanoparticles (Figure 1b). In the FT-IR spectra of GOD−SiO2 and CAT−SiO2 (Figure 1c,d), the peaks at 1543 cm−1 corresponding to the amide II band indicated that the enzymes were successfully immobilized.31

Scheme 1. Schematic Illustration for the Synthesis of Immobilized Enzyme

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GOD−SiO2 maintained more than 85.8% of its initial activity. At pH 3.0, most of free GOD lost activity after 10 h of incubation, while GOD−SiO2 maintained more than 82.4% of its initial activity. These results indicated that the pH stability of GOD was improved significantly after immobilization. The same behavior was also observed for CAT (Figure 3b). The thermal stability of immobilized GOD and CAT was also improved. As shown in Figure 3c, free GOD only retained about 16.6% of its original activity within 10 h at 50 °C, while the immobilized GOD retained more than 83.7% of its original activity under the same condition. Free CAT maintained 21.7% of its original activity within 10 h at 50 °C, while the immobilized CAT retained 48.3% of its original activity (Figure 3d). The enhanced pH and thermal stabilities of GOD−SiO2 and CAT−SiO2 can be ascribed to covalent interactions between the enzyme and support, which can reduce the inactivation of the enzymes under harsh conditions.34 The Michaelis−Menten constants of the free and immobilized enzymes were investigated. As shown in Table 1, the Km value of GOD−SiO2 (7.73 mM) was smaller than that of free GOD (21.75 mM), which could be attributed to the favorable change in the GOD conformation or the decreased enzyme inhibition upon immobilization.35 That is to say, the immobilization process can keep GOD in a more active conformation and then enhanced the binding affinity between GOD and glucose or made the formation of the enzyme− substrate complex easier.35 However, the Km of CAT−SiO2 (358.88 mM) was increased compared to that of free CAT (239.09 mM), which can be explained by the mass transfer limitation and/or reduction in enzyme−substrate affinity.36 3.3. Effect of the Activity Ratio of GOD to CAT on the Removal Efficiency of Glucose. As is well-known, the addition of CAT in excess to the reaction system was done to reduce the inactivation of GOD, since hydrogen peroxide was a substrate of CAT.37 In order to investigate the effect of activity ratio on glucose removal efficiency, an amount of Pickering emulsion which was stabilized by the two enzymes with different activity ratios was added to the reaction system. As shown in Figure 4, the removal efficiency of glucose was increased with the increase of CAT, which indicated that the activity ratio had a significant influence on the removal efficiency. When the activity ratio was 1:4, the highest removal efficiency of 82.53% was obtained, and a further increase of CAT did not improve the removal efficiency obviously. These results indicated that the addition of CAT reduced the H2O2 inhibition, which was similar to the outcome of previous reports.8,37−39 Thus, the activity ratio of 1:4 was adopted for the subsequent experiments. 3.4. Effect of the Dosage of Pickering Emulsion on the Removal Efficiency of Glucose. The dosage of enzyme used is a crucial factor in terms of maximizing economics and process efficiency. In the current study, the increase of the emulsion dosage means the increase of the enzymes dosage. Therefore, the effect of emulsion dosage ranging from 50 to 200 μL on the removal efficiency of glucose was examined, and the results are shown in Figure 5. The removal efficiency was increased by increasing the emulsion dosage from 50 to 190 μL, and the highest removal efficiency was about 88.68% after 6 h when 190 μL of emulsion was used. Further increase of the emulsion dosage did not result in any significant increase of the removal efficiency (Figure 5). These findings could be explained as follows: when the emulsion dosage was below 190 μL, the increase of emulsion dosage would allow for more

Figure 1. FT-IR spectra of the silica (a), APTES-modified silica (b), and GOD−SiO2 (c) and CAT−SiO2 (d) nanoparticles.

The immobilized conditions (including glutaraldehyde concentration, activation time, enzyme concentration, and immobilization time) of GOD−SiO2 and CAT−SiO2 were optimized, and the results are shown in Table S1 of the Supporting Information. The activity recoveries of GOD−SiO2 and CAT−SiO2 were 49.17% and 37.67%, respectively. The reduced activity of the enzymes upon immobilization can be ascribed to the following reason: the enzymes were covalently combined to the supports through Schiff base reactions between aldehyde groups of the support and the amino groups of the lysine residues (ε-amino group) of enzyme molecules, which may result in a distortion of the enzyme structure (i.e., the active site conformation) and partial enzyme inactivation.32,33 On the basis of these results, GOD−SiO2 and CAT− SiO2 used in the subsequent experiments were prepared under the optimal conditions. Most of the oil phases used in the previous studies are organic solvent (i.e., toluene, heptane), which have limited applicability in, for example, pharmaceutical, agricultural, and food fields. Thus, in this study, edible oil (soybean oil) was used as the oil phase. The oil-in-water Pickering emulsion, stabilized by immobilized enzymes, was formed by homogenizing the soybean oil and water in the presence of the GOD− SiO2 and CAT−SiO2 nanoparticles. As shown in Figure 2, the

Figure 2. Optical microscopy images of the Pickering emulsion with different shaking times: (a) 0 day and (b) 15 days.

Pickering emulsion was observed as round spherical droplets, and the droplet size was in the range of 100−250 μm. In addition, the morphology of the Pickering emulsion had little change after 15 days of shaking, which indicated that the stability of Pickering emulsion was excellent. 3.2. The Stability of Free and Immobilized GOD and CAT. In order to evaluate the catalytic properties of the immobilized enzymes, pH and thermal stabilities were studied. As shown in Figure 3a, the activity of GOD−SiO2 decreased more slowly than that of free GOD with the increase of incubation time. For example, after 10 h of incubation at pH 10.0, free GOD only retained 27.0% of the initial activity, while 18165

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Figure 3. pH (a, b) and thermal (c, d) stability of free and immobilized GOD and CAT. Reaction conditions: 25 °C, 10 h.

Table 1. Michaelis−Menten Constants of the Free and Immobilized Enzymes enzyme form

Km (mM)

enzyme form

Km (mM)

free GOD GOD−SiO2

21.75 7.73

free CAT CAT−SiO2

239.09 358.88

Figure 5. Time profiles on the removal efficiency of glucose in different amounts of Pickering emulsion. Reaction conditions: 1:4 activity ratio of GOD to CAT, 5 mL of IMO (500 mg/mL), 30 °C, pH 6.0, 6 h.

of glucose increased gradually with temperature up to an optimum temperature of 45 °C and then decreased. According to the Arrhenius equation, reaction rate increases with the increase of temperature.42 However, this principle was complied to up to 45 °C, and over 45 °C the removal efficiency decreased. Deactivation of enzymes caused by high temperature may be one of the main factors for the drop of removal efficiency. Since enzyme activity is dependent on the ionization state of the amino acids in the active site, pH plays an important role in maintaining the proper conformation of an enzyme.18 As shown in Figure 6b, the removal efficiency of glucose increased as the pH increased and then decreased. The highest removal efficiency of 97.3% was obtained at pH 6.0, which may be attributed to the optimal pH of the used GOD being 6.0. On the basis of the above results, a temperature of 45 °C and pH 6.0 were adopted for the subsequent experiments. 3.6. Recycling Stability of the Pickering Emulsion. Another important issue for the potential of the system was the recycling stability. Thus, the recycling stability of this Pickering

Figure 4. Time profiles of the removal efficiency of glucose in different activity ratios of GOD to CAT. Reaction conditions: GOD activity of 12.44 U, 100 μL of Pickering emulsion, 5 mL of IMO (500 mg/mL), 30 °C, pH 6.0, 6 h.

substrate molecules to be absorbed by the enzyme active site40 and then improved the reaction rate and the removal efficiency. When the emulsion dosage was above 190 μL, further increase in the emulsion quantity did not have much effect on the removal efficiency. This leveling-off phenomenon at higher enzyme quantity was typical, and the results were consistent with those of previous reports using other enzymes.24,41 Thus, to minimize the cost of the purification process, 190 μL of Pickering emulsion was adopted in the subsequent experiments. 3.5. Effects of Reaction Temperature and pH on the Removal Efficiency of Glucose. The variation of reaction temperature has a considerable effect on the removal efficiency of glucose. As can be seen in Figure 6a, the removal efficiency 18166

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Figure 6. Time profiles on the removal efficiency of glucose at different temperatures (a) and pH values (b). Reaction conditions: GOD activity of 23.64 U, 1:4 activity ratio of GOD to CAT, 190 μL of Pickering emulsion, 5 mL of IMO (500 mg/mL), 6 h.

purity IMO on an industrial scale. Additionally, magnetic nanocatalysts can be separated simply and efficiently from reaction mixtures with an external magnetic field and have been used in many reactions.44 Introducing magnetic nanoparticles to immobilize the GOD−CAT system may be another good alternative to prepare highly efficient biocatalysts for IMO purification.

emulsion system was determined by its repeated use for removing glucose. The Pickering emulsion can quickly float to the air/water interface soon after the shaking stopped, which ensured that this Pickering emulsion was easily recyclable.25 Upon the completion of one batch, the Pickering emulsion was collected and used for the next batch. From Figure 6a,b, all reactions reached a final plateau after 3 h. Thus, in the tests of recycling stability, the reactions were run for 3 h. For comparison, the simply mixed immobilized enzymes (denoted “nanoparticles-system”) were also used to remove glucose. As shown in Figure 7, a decrease of removal efficiency was

4. CONCLUSIONS In this study, GOD and CAT were successfully immobilized on silica nanoparticles, and the properties of the enzymes were improved. For removing glucose from IMO, a Pickering emulsion stabilized by the immobilized GOD and CAT nanoparticles (a bienzymatic system) was constructed. The reaction parameters (e.g., activity ratio of GOD to CAT, the amount of Pickering emulsion, temperature, and pH) for the removal of glucose were optimized. The results indicated that about 97.3% of glucose can be removed under the optimal conditions. After recycling five times, the removal efficiency can still remain at about 73.57%. Additionally, the generated gluconic acid can be removed through precipitation by the addition of Ca(OH)2. Therefore, this bienzymatic process is ideal as an auxiliary process to the enzymatic process for the removal of glucose from IMO. The present enzyme-stabilized Pickering emulsion will be able to evolve as a platform technology to construct novel and efficient multienzymatic systems for a variety of applications.

Figure 7. Recycling stability of the Pickering emulsion and nanoparticles-system. Reaction conditions: GOD activity of 23.64 U, 1:4 activity ratio of GOD to CAT, 190 μL of Pickering emulsion, 5 mL of IMO (500 mg/mL), 45 °C, pH 6.0, 3 h.

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observed as the recycling times increased. The decline in removal efficiency may be ascribed to enzyme inactivation and enzyme loss in the reaction process.26 After five reaction cycles, the removal efficiency of the Pickering emulsion system remained about 73.57%, while that of the nanoparticles-system remained about 62.32%. This can be attributed to the easy separation of the Pickering emulsion system, which reduced the mass loss of the biocatalyst during the centrifugation process (for nanoparticles-system) after each reaction batch.43 On the basis of the above results, the advantages of this method are as follows: (1) glucose can be removed efficiently because of the substrate specificity of GOD; (2) the Pickering emulsion can be reused several times without appreciable loss of its activity; (3) when stirring or shaking was stopped, the Pickering emulsion can float onto the air/water interface quickly and then make the separation process easy; and (4) the byproduct of gluconic acid can be separated easily by precipitation and can also find applications in the pharmaceutical and food industries. Thus, there is obviously a great potential in using this Pickering emulsion system as biocatalyst to remove glucose and then to facilitate the production of high-

ASSOCIATED CONTENT

S Supporting Information *

Characterization of the silica nanoparticles and Pickering emulsion, activity assays of GOD and CAT, and Table S1. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-22-60204945. Fax: +86-22-60204294. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Nature Science Foundation of China (Nos. 21276060, 21276062 and 21306039), the Natural Science Foundation of Tianjin (13JCYBJC18500), and the Science and Technology Research Key Project of Higher School in Hebei Province (YQ2013025). 18167

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