Horseradish Peroxidase Immobilized on Multifunctional Hybrid

Jun 21, 2019 - The microspheres before and after enzyme immobilization were dried and .... The AFB1 retention time was around 25 ± 1 min, and the AFB...
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Article Cite This: Ind. Eng. Chem. Res. 2019, 58, 11710−11719

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Horseradish Peroxidase Immobilized on Multifunctional Hybrid Microspheres for Aflatoxin B1 Removal: Will Enzymatic Reaction be Enhanced by Adsorption? Fangfang Zhou,†,‡ Jianquan Luo,*,†,‡ Benkun Qi,† Xiangrong Chen,† and Yinhua Wan†,‡ †

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State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China ‡ School of Chemical Engineering, University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China S Supporting Information *

ABSTRACT: As the most dangerous carcinogens, efficient and green removal of Aflatoxin B1 (AFB1) is imperative. For the first time, horseradish peroxidase (HRP) was immobilized in/on an alginate/chitosan/montmorillonite (SA/CS/MON) hybrid microsphere and applied for the AFB1 removal. Compared to the enzyme immobilization via encapsulation and adsorption followed by cross-linking, HRP immobilized on the microsphere surface via covalent bonding showed the highest specific activity and AFB1 removal efficiency. The negatively charged MON could not only adsorb abundant AFB1 but also attract more CS chains on the microsphere surface and bring more active sites for enzyme immobilization and cross-linking; thus, the SA/CS/MON microspheres had stronger antiswelling ability and higher AFB1 adsorption capacity and enzyme loading. Although the AFB1 enrichment in the hybrid microsphere did not promote its degradation by enzymatic catalysis, the HRP-loaded hybrid microsphere could be reused and the enzymatic degradation of AFB1 increased a little as the adsorption became saturated. The enzymatic reaction was not enhanced by the substrate adsorption in this case because the AFB1 adsorption by the hybrid microspheres would decrease the AFB1 concentration in bulk solution and the degradation products might be adsorbed. The adsorbent (i.e., matrix for enzyme immobilization) has different adsorption ability to the substrate/product, and a catalyst with high activity on the substrate is desirable to maximize the synergistic effect of adsorption and catalysis. The obtained hybrid microsphere prototype is promising as a multifunctional matrix for many purposes since both the adsorbent and the catalyst can be designed.

1. INTRODUCTION Aflatoxins (AFs) are toxic secondary metabolites produced by Aspergillus f lavus and Aspergillus parasiticus. Due to its widespread pollution, AF has become one of the key hazardous factors that cause serious contamination to crop, food, and feed.1 Aflatoxin B1 (AFB1), as the most toxic mycotoxin, was classified as a human carcinogen (Group 1) by the International Agency for Research on Cancer (IARC) in 1993.2 AFB1 could contaminate food crops under appropriate conditions of temperature and humidity at harvest or postharvest stages. In order to conserve resources, the contaminated grains are fermented to produce alcohol for biofuel, and vinasse produced by fermentation is widely used for animal feed. However, it has been confirmed that AFB1 does not degrade during the fermentation process and is enriched in vinasse, which causes a severe threat to animal health.3 There are many methods used to remove AFB1, such as adsorption,4 radiation,5 ammonia/ozone,6 and microbial and enzymatic degradation. AFB1 adsorption by clays and © 2019 American Chemical Society

microbial cells is highly efficient, but the collection and reuse of the adsorbents are difficult, and the AFB1 desorption from the adsorbents under certain conditions is inevitable.7,8 Enzymatic degradation is a green process for AFB1 removal and does not produce secondary pollution. Laccase,9 manganese peroxidase,10,11 and aflatoxin-oxidase have been successfully applied to degrade AFB1. Compared with these enzymes, horseradish peroxidase (HRP) has the advantages of easy availability and high specific activity, which has been widely used in catalytic oxidation of aromatic compounds.12 Like other enzymes, its degradation efficiency is relatively low and it easily loses activity with lengthening of time, especially at high temperature and/or acidic/alkaline pH. The stability and reusability of enzymes can be improved by immobilizing enzymes in/on the matrix via physical Received: Revised: Accepted: Published: 11710

April 17, 2019 June 20, 2019 June 21, 2019 June 21, 2019 DOI: 10.1021/acs.iecr.9b02094 Ind. Eng. Chem. Res. 2019, 58, 11710−11719

Article

Industrial & Engineering Chemistry Research

Figure 1. Schematic diagrams of (a) preparation of SA/CS/MON microspheres and (b) HRP immobilization on the microsphere via covalent bonding.

AFB1 removal efficiency. For example, He et al. applied rice husk carriers to immobilize zearalenone-degrading enzyme to remove zearalenone (one of mycotoxin) and claimed that the adsorption and degradation efficiencies were 28% and 65%, respectively.20 Theoretically, if the reactant is adsorbed in/on the immobilization matrix, such reactant enrichment effect may increase the catalytic efficiency of the immobilized catalyst. Ma et al. reported that polydopamine carriers dramatically enhanced the catalytic performance (∼450%) of Au nanoparticles in methylene blue reduction as a result of the adsorption of reactants by polydopamine.21 Gkaniatsou et al. found that the oxidation rate of methyl orange by microperoxidase-8 was significantly increased after enzyme immobilization on metal−organic framework MIL-101(Cr). The preconcentration of the methyl orange reactant on the immobilization carrier was most likely responsible for its enhanced degradation.22 Therefore, it is speculated that introduction of MON into Ca−Alg microsphere for enzyme immobilization can promote the enzymatic degradation of AFB1. In this study, we attempted to fabricate a novel organic/ inorganic hybrid microsphere for immobilization of HRP and removal of AFB1. As illustrated in Figure 1, a mixed solution of sodium alginate (SA) and montmorillonite (MON) is dropwise added into chitosan−calcium chloride (CS−CaCl2) solution to obtain SA/CS/MON microspheres (chitosan, CS, was added in order to increase the stability and functional groups on the microspheres), which are used for immobilization of HRP via entrapment, adsorption/cross-linking, and covalent bonding. These enzyme-loaded microspheres with adsorption and catalytic functions were applied for AFB1 removal, and the physicochemical characterizations, enzyme immobilization behaviors, stability, reusability, and AFB1 removal efficiency of the SA/CS microspheres with and without MON were compared, where the effect of the MON

adsorption/entrapment and covalent bonding. Besides the immobilization protocol, the matrix is another important factor affecting the performance of immobilized enzyme. Many matrices have been applied to immobilize enzymes, such as polymeric/ceramic membranes,13,14 microspheres,15 nanomaterials,16 mesoporous,17 and natural materials. Among these, calcium alginate (Ca−Alg) microsphere is one of the most popular matrixes for enzyme immobilization because of its easy preparation and good biological compatibility. Enzymes can be entrapped into Ca−Alg microspheres during preparation or be covalently linked on the microspheres surface after functionalization. However, the resultant Ca−Alg microspheres always exhibit unsatisfactory structural stability due to serious swelling in aqueous solutions, thus causing enzyme leakage and sphere damage. Great efforts have been devoted to improving the stability of Ca−Alg microspheres. Wang et al. modified alginate with dopamine, and the resultant microspheres were reinforced by self-polymerization of dopamine and cross-linking with calcium ions.18 Ai et al. prepared boehmite/alginate hybrid microspheres, and the better stability due to the electrostatic interaction between alginate and boehmite ensured “zero enzyme leaching” after incubation in Tris buffer for 67 h.19 Therefore, the organic/inorganic hybrid microspheres showed an improved performance for enzyme immobilization. Inspired by these reports, in the present work, montmorillonite (MON), as one of the best inorganic absorbents for AFB1, was introduced into Ca−Alg microsphere.1 We hypothesize that the obtained hybrid microspheres would not only own better antiswelling ability and stronger adsorption capacity of AFB1 than Ca−Alg microspheres but also enable the easy collection and reuse of the clay absorbents. Moreover, when oxidase is immobilized in/on the matrix with AFB1 adsorption ability, the combination of physical adsorption and enzymatic degradation may greatly enhance the 11711

DOI: 10.1021/acs.iecr.9b02094 Ind. Eng. Chem. Res. 2019, 58, 11710−11719

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Industrial & Engineering Chemistry Research

enzyme immobilization and the addition of MON via an FTIR spectrometer (Nicolet iS50, USA). 2.4.3. Swelling Studies. Accurately weighed microspheres were immersed in 20 mL of acetate buffer (10 mM, pH 5.0) at 50 °C and taken out at specific time intervals. They were blotted with filter paper to absorb water on the surface and then weighed immediately.26 Swelling degree (SD) of the sample was calculated according to the following expression

adsorption of AFB1 on the catalytic efficiency of the immobilized HRP was emphasized. The outcomes of this work not only offer a green alternative for the mycotoxin removal from animal feed but also clarify the interplay between adsorption and catalysis of the enzyme-loaded microsphere.

2. EXPERIMENTAL SECTION 2.1. Materials. CS (deacetylation degree ≥ 95% and viscosity of 100−200 mPa.s) and SA (viscosity of 200 ± 20 mPa.s) were purchased from Macklin, China. MON was provided by Sigma-Aldrich. AFB1 (98%) from Aspergillus f lavus was purchased from J&K Scientific, China. Peroxidase (EC 1.11.1.7, >300 units·mg−1) from horseradish and 4aminoantipyrine were purchased from Aladdin. Bovine serum albumin (BSA, 97%) was provided by Solarbio, China. All other chemical reagents were commercial analytical grade and used without further purification. Acetate buffer solution (pH = 5, 10 mM) was used for preparation of enzyme and AFB1 solution. 2.2. Preparation of SA/CS and SA/CS/MON Microspheres. A 2% SA solution was prepared by dissolving 2.0 g of SA in 100 mL of deionized water. The CS−CaCl2 mixed gelling solution was prepared as follows: 2.22 g of CaCl2 and 1 g of CS were dissolved in 100 mL of 2% acetic acid solution. A 5 mL amount of SA solution with or without MON (5 g/L) was dropped into 20 mL of mixed gelling solution through a 0.45 mm syringe needle at a dropping rate of 0.1 mL/min. The microspheres were cured for 30 min in the gelling solution at room temperature with gentle stirring, and then the microspheres were filtered and rinsed with deionized water.23 2.3. HRP Immobilization. 2.3.1. Encapsulation. A 3 mg amount of free HRP was added into 5 mL of 2% SA solution, and the enzyme-loaded microsphere was prepared according to the procedures described in section 2.2. The obtained microspheres were then cross-linked in 2% GA solution for 1 h, and the enzyme-loaded microspheres were filtered and washed with deionized water.24,25 2.3.2. Adsorption/Cross-Linking. The SA/CS microspheres prepared in section 2.2 were dispersed in 0.15 g/L HRP solution (3 mg of free HRP was added into 20 mL of acetate buffer solution), gently stirred for 2 h, filtered, and cross-linked with 2% GA for 1 h. 2.3.3. Covalent Bonding. The SA/CS and SA/CS/MON microspheres were activated in 2% GA for 1 h at room temperature, and then the microspheres were thoroughly washed with deionized water. After that the activated microspheres were kept in the solution of HRP for 2 h with a concentration of 0.15 g/L. All of the enzyme-loaded microspheres were washed thoroughly and stored in phosphate buffer (10 mM, pH = 7.0) at 4 °C. 2.4. Characterization. 2.4.1. Scanning Electron Microscope (SEM) and Energy-Dispersive X-ray Spectroscopy (EDX). The microspheres before and after enzyme immobilization were dried and coated with gold under reduced pressure. Their morphology was examined using a JSM-6700F Field Emission SEM (SU8020, Hitachi, Japan) in cross section (5 kV operational voltages) and perform EDX measurements (20 kV operational voltages). EDX measurements were obtained between 0 and 20 keV. 2.4.2. Fourier Transform Infrared Spectroscopy (FTIR). FTIR spectra were tested for the SA/CS, SA/CS/MON, and SA/CS/MON-HRP microspheres to verify the successful

SD( %) =

W − W0 × 100% W0

where W is the weight of the swollen microspheres and W0 is the initial weight of the microspheres. 2.5. Enzyme Activity Measurement. The catalytic activity of HRP was measured using a mixture of phenol, 4aminoantipyrine, and H2O2 as substrates. Phenol and 4aminoantipyrine were dissolved in acetate buffer (10 mM, pH 5.0) to prepare the substrate solution, in which their concentrations were 40 and 4 mM, respectively. Subsequently, free enzyme or immobilized enzyme was added into a 20 mL substrate solution under continuous magnetic stirring. After 0.5 mM H2O2 was added, the absorbance change of the solution at 505 nm caused by phenol and 4-aminoantipyrine consumption was recorded. One unit of catalytic activity (U) is defined as the enzyme amount consuming 1 μmol of H2O2 per minute under the assay conditions (25 °C, pH 5.0).13 Enzyme specific activity was calculated according to the following equation specific activity (U/mg) =

catalytic activity (U) enzyme amount (mg)

The thermal and storage stabilities of the immobilized HRP were evaluated by the following procedures. Briefly, the thermal stability was investigated by measuring the residual activity of the immobilized HRP after having been incubated at 50 °C in a 10 mM acetate buffer (pH 5.0) for different times (1, 2, 3, 4 h). The storage stability was determined through selectively measuring the residual activity of the immobilized HRP after having been stored for 1, 2, 3, 6, 9, and 12 days at 4 °C, respectively. For all stability experiments, the initial activity of the immobilized HRP is assumed as 100%, while other activities are the relative values compared with the initial activity. Each result was obtained by averaging three individual experiments. 2.6. AFB1 Adsorption Isotherm. The AFB1 adsorption isotherm with the SA/CS and SA/CS/MON microspheres was investigated (more details of the analytical procedures and data processing are described in the Supporting Information). 2.7. AFB1 Removal. 2.7.1. AFB1 Removal by H2O2 Oxidation. Different concentrations of H2O2 (final concentration 0.01−4 mM) were dispersed in 20 mL of AFB1 solution (100 ppb, 10 mM acetate buffer, pH 5.0) at 50 °C and 150 rpm, and 1 mL of solution was taken out for analysis at different time intervals. 2.7.2. AFB1 Removal by Adsorption. A 0.3 g amount of HRP-loaded microspheres (without H2O2) was dispersed in 20 mL of AFB1 solution (100 ppb, 10 mM acetate buffer, pH 5.0) at 50 °C and 150 rpm, and 1 mL of solution was taken out for analysis at different times. 2.7.3. AFB1 Removal by Enzymatic Catalysis. For free enzyme, 3 mg of HRP and 0.5 mM H2O2 were added into 20 mL of AFB1 solution (100 ppb, 10 mM acetate buffer, pH 5.0) 11712

DOI: 10.1021/acs.iecr.9b02094 Ind. Eng. Chem. Res. 2019, 58, 11710−11719

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Figure 2. Photographs of microspheres: (A) SA/CS microspheres, (B) SA/CS/MON microspheres, (C) SA/CS/MON-HRP microspheres. Surface SEM images of microspheres: magnification from left to right is ×50, ×1000, ×3000.

Figure 3. EDX spectra of SA/CS/MON-HRP microspheres. (a) EDS layered image. (b) Electronic image. (c, d, and e) Distributions of the elements Si, Al, and S on the surface of SA/CS/MON-HRP microspheres, respectively. (g) Surface spectrum.

0.22 μm syringe filter before HPLC analysis. The AFB1 retention time was around 25 ± 1 min, and the AFB1 detection limit in this protocol was 5 μg/L.9 2.8. Reusability. The reusability of the HRP-loaded microspheres was determined by measuring the AFB1 removal with reuse cycle. After the first reaction cycle as described in section 2.7.3 (H2O2 concentration was 0.1 mM), the HRPloaded microspheres were collected after 2 h reaction and then reused with fresh reactants for the next cycle. Each experiment was repeated for three times. 2.9. Identification of AFB1 Degradation Products. Mass spectrometry (MS) analysis was carried out to analyze the AFB1 degradation products27 (more details of the analytical procedures are described in the Supporting Information).

for 2 h at 50 °C and 150 rpm. For immobilized HRP, 0.3 g of HRP-loaded microspheres and different H2O2 concentrations (final concentration 0.01−4 mM) were dispersed in 20 mL of AFB1 solution (100 ppb, 10 mM acetate buffer, pH 5.0) at 50 °C and 150 rpm, and 1 mL of solution was taken out for analysis after a period of time catalytic efficiency (%) = E0 (%) − E1 (%) − E2 (%)

where E0 is the total removal efficiency, E1 is AFB1 removal by H2O2 oxidation, and E2 is AFB1 removal by adsorption. The substrate inhibition kinetics of the HRP-catalyzed AFB1 degradation was measured at different AFB1 concentrations (50−1000 ppb) with free HRP of 0.05 g/L and 0.1 mM H2O2. AFB1 concentration was measured by high-performance liquid chromatography (HPLC) (Agilent, U.S.A.) installed with a fluorescence detector (FLD) (Agilent, U.S.A.) and a C18 HPLC column (ZORBAX SB-C18, 250 mm × 4.6 mm i.d.; 5 μm; Agilent, U.S.A.). A 45% methanol aqueous solution was used as the mobile phase for HPLC analysis at a flow rate of 0.8 mL/min. AFB1 is detected by FLD with an excitation wavelength of 365 nm and an emission wavelength of 440 nm. The injection volume was 50 μL, and the temperature of the column oven was 30 °C. All samples were pretreated using a

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of Microspheres. The SA/CS microspheres were fabricated by dropwise adding the SA into a CS−CaCl2 mixed solution. The optical images (Figure 2A, 2B, and 2C) demonstrate that the SA/CS microspheres are translucent, while the SA/CS/MON microspheres are dark yellow, which can be regarded as an indication 11713

DOI: 10.1021/acs.iecr.9b02094 Ind. Eng. Chem. Res. 2019, 58, 11710−11719

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was successfully covalently immobilized on the surface of the SA/CS/MON microspheres.30,37 3.2. HRP Immobilization and AFB1 Removal by SA/CS Microspheres. As shown in Figure 5A, when HRP is immobilized by different strategies, the SA/CS-HRP microspheres prepared by encapsulation have the highest enzyme loading of 2.36 mg/g, while it is around 1.07 mg/g using covalent bonding strategy. The SA/CS-HRP microsphere prepared by adsorption/cross-linking has the lowest enzyme loading, only 0.41 mg/g. As shown in Figure 5B, the storage stability of the SA/CS-HRP microspheres prepared by encapsulation is the best, and 70% of its initial activity can be maintained after 6 days. After 12 days, the enzyme activity of the SA/CS-HRP microspheres prepared by adsorption/ cross-linking or covalent bonding is almost completely lost. It is reasonable that encapsulation can perfectly maintain HRP conformation and alleviate enzyme leakage caused by the SA/ CS dissolution. However, the enzyme immobilized by covalent bonding shows the highest specific activity, though its enzyme loading is much lower than that using the encapsulation method (Figure 5C), which is caused by the difference in mass transfer resistance of substrate to the enzymes immobilized via two methods, that is, the immobilized enzymes on the microsphere surface via covalent bonding are more accessible to the substrate. After 1 h soakage at 50 °C, the specific activity of the immobilized enzyme by both encapsulation and covalent bonding increases obviously, which is caused by the microsphere swelling decreasing the mass transfer resistance for the encapsulated enzyme and increasing the reaction interface area for the immobilized enzyme on the microsphere surface. While for the SA/CS-HRP microspheres prepared by adsorption/ cross-linking, the swelling is limited and the enzyme density on the microsphere is the lowest, and that is why the sudden increase of the specific activity with soaking time does not occur. Regarding the AFB1 removal (Figure 5D), H2O2 alone can achieve 7.5% by chemical oxidation, and free HRP is able to degrade 9.63% of AFB1 by enzymatic catalysis, while the SA/CS microsphere also adsorbs 4.5% of AFB1. Subsequently, we immobilized HRP on the SA/CS microspheres by different strategies and explored the removal efficiency of AFB1 when the microsphere adsorption and enzyme catalysis existed simultaneously. It was found that the AFB1 removal using the SA/CS-HRP microspheres prepared by encapsulation and adsorption/cross-linking did not significantly increase compared to that by the mixture of SA/CS microsphere and H2O2, indicating that their immobilized HRP has almost no degradation effect on AFB1. Only the immobilized HRP by covalent bonding displays an obvious enzymatic degradation of AFB1, around 7%, because it has the highest activity (Figure 5C) and the least mass transfer resistance between enzyme and AFB1. Compared with the sum of the AFB1 removal by the microsphere adsorption and free HRP catalysis (14%), the immobilized HRP by covalent bonding still shows higher AFB1 removal (19%), implying that enzyme immobilization on microsphere can improve the catalytic ability of HRP on AFB1. Therefore, the covalent bonding method was applied for HRP immobilization in the following studies. 3.3. Inorganic/Organic SA/CS/MON Microspheres. In order to increase AFB1 removal, MON with strong adsorption capacity to AFB1 was introduced into the SA/CS microsphere. As shown in Figure 6A, the SA/CS/MON microspheres have lower swelling degree than the SA/CS microspheres, which is caused by the intertwining between the flexible SA molecule

of the successful inclusion of MON. The SA/CS/MON-HRP microspheres seem pink, verifying that HRP is successfully immobilized on the SA/CS/MON microspheres. The surface microstructure of microspheres after freeze drying was observed by SEM, and as seen in Figure 2, all of the dried microspheres are uniform in size, which is about 2 mm in diameter. However, there are some cracks caused by partial collapsing of the polymer network during dehydration. The surface of the SA/CS microspheres is rough, and there are serious wrinkles. After addition of MON, the surface of the SA/CS/MON microspheres becomes smoother and their mechanical strength increases. In addition, the SA/CS/MON microspheres remain relatively complete spherical (Figure 2b1), and the grafting of HRP further reduces their surface roughness (Figure 2c1). The morphology of our freeze-dried microspheres is similar to those reported in the literature.19,28,29 MON is 2:1 phyllosilicate mineral with a large amount of silicon (Si) and aluminum (Al), while HRP is a protein containing sulfur (S) elements different from SA, CS, and MON. It was reported that materials elements including S element in enzyme molecules could be detected by EDX.30 As shown in Figure 3, the presence of Si, Al, and S elements further confirms the integration of MON and HRP in/on the microspheres. The FTIR spectrum of SA/CS microspheres (Figure 4) shows a broad band at 3418 cm−1 assigned to the overlapping

Figure 4. FTIR spectra of SA/CS, SA/CS/MON, and SA/CS/MONHRP microspheres

of O−H and N−H stretching. The band at 1642 cm−1 is attributed to the CO stretching (amide I), and a band of weak intensity near 1540 cm−1 is assigned to the N−H bending (amide II).31 The band at 1421 cm−1 corresponds to the symmetric stretching vibrations of carboxyl (COO−) groups of alginate molecules.32,33 The bands at 2887 and 1035 cm−1 are ascribed to the C−H stretching and to the skeletal vibration involving CO stretching, respectively, which are also characteristic peaks of the saccharide structure.25,34 In addition, the above bands associated with the SA/CS microspheres remain unaltered in the FTIR spectrum of SA/ CS/MON microspheres, but a suspended aldehyde peak appears at 2720 cm−1 and a Si−O−Si occurs at 527 cm−1.35,36 The band intensity of SA/CS/MON-HRP microspheres at 1642 and 1540 cm−1 increases to varying degrees due to the formation of a large number of amide bonds between the amino of HRP and the aldehyde of GA, confirming that HRP 11714

DOI: 10.1021/acs.iecr.9b02094 Ind. Eng. Chem. Res. 2019, 58, 11710−11719

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Figure 5. Comparison of three enzyme immobilization strategies with SA/CS microspheres: (A) Enzyme loading, (B) storage stability (4 °C, 10 mM phosphate buffer, pH 7.0), (C) thermostability (50 °C, 10 mM acetate buffer, pH 5.0), and (D) AFB1 removal efficiency.

Figure 6. (A) Swelling degree (50 °C, 10 mM acetate buffer, pH 5.0) with storage time, and (B) enzyme loading of the SA/CS and SA/CS/MON microspheres using covalent bonding strategy.

chains and the rigid MON particles (MON limits the movement of the SA chains and then increases the rigidity of the microsphere).38−40 Moreover, the AFB1 adsorption isotherm with the SA/CS and SA/CS/MON microspheres shows that their maximum adsorption capacities are 1.2018 and 43.4782 μg/g, respectively, and the heterogeneity of adsorption sites and multilayer adsorption for AFB1 exists in the SA/CS/MON microspheres (Figure S1 and Table S1). The inclusion of MON in the microsphere also improves the enzyme loading by 16.8% using a covalent bonding strategy, as shown in Figure 6B. This can be explained by the fact that MON (layered mineral) with negative charge adsorbs more positively charged CS with amino groups during microsphere preparation, resulting in more aldehyde groups on the microsphere surface after GA activation for HRP bonding. 3.4. Thermostability. As shown in Figure 7, the specific activities of the SA/CS and SA/CS/MON microspheres increase 2-fold when incubated at 50 °C for 2 h, which is caused by the enhancing reaction interface area due to microsphere swelling. However, with increasing incubation time from 2 to 10 h, their specific activity is decreasing owing

Figure 7. Thermostability of the SA/CS-HRP and SA/CS/MONHRP microspheres (50 °C, 10 mM acetate buffer, pH 5.0).

to the possible enzyme leakage and inactivation with time. It was found that the SA/CS/MON-HRP microspheres can better keep the enzyme activity probably because the more CS chains with amino groups surrounding the microspheres 11715

DOI: 10.1021/acs.iecr.9b02094 Ind. Eng. Chem. Res. 2019, 58, 11710−11719

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Figure 8. Effect of H2O2 concentration (A) on the SA/CS-HRP and (B) SA/CS/MON-HRP microspheres in the removal of AFB1.

Figure 9. Proportion of AFB1 removal efficiency of by (A) SA/CS-HRP and (B) SA/CS/MON-HRP microspheres during the continuous reuse of the microspheres, partial removal of AFB1 due to (C) adsorption, and (D) enzymatic catalysis with increasing reuse cycle.

brought by MON provide more cross-linking sites, alleviating the enzyme leakage and improving enzyme stability. 3.6. Effect of H2O2 Concentration. As shown in Figure 8, with an increase of H2O2 concentration, the chemical oxidation of AFB1 gradually enhances while the AFB1 adsorption by the microspheres does not change. For the enzymatic degradation of AFB1, it first elevates when H2O2 concentration increases from 0.01 to 0.1 mM and then becomes lower with further increasing H2O2 concentration, especially for the SA/CS/ MON-HRP microspheres (the enzymatic contribution on the AFB1 removal almost disappears when H2O2 concentration is more than 1 mM). This implies that there is a substrate inhibition effect on HRP for AFB1 removal, and since the SA/ CS/MON-HRP microspheres may adsorb more AFB1 and H2O2, such inhibition effect is more important. Therefore, a final H2O2 concentration of 0.1 mM is used for the following studies. Regarding the AFB1 degradation products catalyzed by HRP, MS analysis was carried out and it was found that the lactone and bifuran rings (main toxic structural components) of AFB1 were destroyed (Figures S2 and S3). 3.7. Reusability of Enzyme-Loaded Microsphere. From the above results it is found that the enzymatic

contribution on the AFB1 removal is not significant as expected, especially for the SA/CS/MON-HRP microsphere. We speculated that if the adsorption capacity of the microsphere became saturated with reuse cycle, the contribution of enzymatic catalysis on the AFB1 removal might be enhanced. However, as seen in Figure 9, the AFB1 removal by both microspheres decreases gradually after the second reuse cycle (without regeneration elution). Moreover, the contribution of adsorption to the AFB1 removal is decreasing because more and more adsorption sites become unavailable (Figure 9C), while the enzymatic catalysis on the AFB1 removal only increases a little for the SA/CS/MON-HRP microsphere (Figure 9D). In detail, the AFB1 removal by adsorption for the SA/CS/MON-HRP microsphere decreases from 75% to 50%, while it reduces from 6.5% to 0% for the SA/CS-HRP microsphere, which can be explained by their different adsorption capacity (Figure 9C). For the AFB1 removal by enzymatic catalysis, the SA/CS -HRP microsphere does not show an obvious change in the first four reuse cycles (15%), but there is a decline in the fifth reuse cycle (this may be caused by a decrease of enzyme activity, as shown in Figure 7); the SA/CS/MON-HRP microsphere has an increasing trend 11716

DOI: 10.1021/acs.iecr.9b02094 Ind. Eng. Chem. Res. 2019, 58, 11710−11719

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which cannot contact the HRP immobilized on the surface by covalent bonding. Consequently, effect of MON proportion in microspheres on AFB1 removal by the immobilized HRP via encapsulation was studied. As shown in Figure 11B, the enhancement effect by the AFB1 adsorption on the enzymatic catalysis is still not obvious even though the HRP is mixed with MON in the microsphere. We can conclude that when HRP is immobilized, the mass transfer resistance increases and the AFB1 affinity to the active center decreases, and thus, the substrate enrichment by adsorption around the immobilized HRP is not helpful to the degradation efficiency. It is possible that the adsorption ability to the degradation product also inhibits the catalytic performance, and therefore, the adsorbent (i.e., matrix for enzyme immobilization) who has different adsorption ability to the substrate/product and the catalyst with high activity on the substrate are desirable to maximize the synergistic effect of adsorption and catalysis. The multifunctional microsphere with immobilized HRP shows a higher AFB1 removal than the microsphere adsorbent, and with the same mass of microspheres, the AFB1 degradation capacity of the SA/CS/MON-HRP microspheres is 17.25% higher than that of the SA/CS/MON microspheres. It is worth mentioning that such microsphere will be more valuable for removing multimycotoxin when both adsorption and enzymatic degradation are indispensable.

from 6.5% to 15% until the fourth reuse cycle (Figure 9D), implying that too much AFB1 adsorbed in the microsphere would inhibit the enzymatic degradation of AFB1, and the decrease in AFB1 removal by adsorption (from 75% to 50%) can promote the enzymatic catalysis of AFB1. There are two possible explanations: the first is that the high adsorption capacity of the microspheres leads to the decrease of substrate concentration in the bulk solution, which reduces the contact possibility of AFB1 molecules with the immobilized HRP on the microsphere surface; the second is that the adsorption causes substrate enrichment on the microsphere surface, resulting in a substrate inhibition effect on the immobilized HRP. It can be seen that in Figure 10 when the AFB1

4. CONCLUSIONS Although enzyme loading and storage stability of HRP immobilized in the SA/CS microsphere via encapsulation are the highest, enzyme immobilization on the microsphere surface via covalent bonding is considered the most suitable for AFB1 removal because mass transfer is the most important to HRP activity on the AFB1 degradation. Inclusion of MON into the SA/CS microsphere greatly improves the antiswelling ability, AFB1 adsorption capacity, enzyme loading, and stability, but such strong AFB1 adsorption by the SA/CS/MON microsphere does not promote the AFB1 removal by enzymatic catalysis and even shows some inhibition effect. The AFB1 adsorption would decrease the AFB1 concentration in bulk solution, and the degradation products may also be adsorbed, thus overcoming the possible positive effect produced by the substrate enrichment. When the HRP-loaded hybrid microspheres are reused without regeneration, their catalytic ability increases as the adsorption becomes saturated but the maximum degradation rate is only 15%. Introduction of MON into the SA/CS microsphere improves the AFB1 adsorption more than 10 times, and the immobilized HRP on

Figure 10. Effect of AFB1 concentrations on its enzymatic degradation rate by free HRP.

concentration is higher than 800 μg/L (8 times of the feed solution), the AFB1 degradation efficiency starts to decline. Even though the adsorption of AFB1 decreases to zero, the AFB1 removal by enzymatic catalysis is only 15% (Figure 9D), indicating that AFB1 is really refractory and more efficient enzyme for AFB1 degradation is required. As shown in Figure 10, when the AFB1 concentration is less than 700 μg/L, the AFB1 degradation catalyzed by HRP increases with the AFB1 concentration. We speculated that if the AFB1 adsorption amount was manipulated by changing the MON proportion in the microsphere, the positive effect of AFB1 adsorption on the enzymatic catalysis might occur. However, as seen in Figure 11A, when 0.01% MON is added into the SA solution, the AFB1 removal by enzymatic catalysis is suppressed, although the AFB1 adsorption is only 10%. We may argue that AFB1 would be adsorbed into the microsphere,

Figure 11. Effect of MON proportion in microspheres on AFB1 removal by adsorption and enzymatic catalysis, respectively, for enzyme immobilization via (A) covalent bonding and (B) encapsulation strategies: 0.01, 0.05, 0.2, 0.5 g of MON was added into 100 mM SA solution. 11717

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the SA/CS/MON microspheres can further increase the AFB1 removal by 17.25%. The obtained hybrid microsphere as a prototype is promising in the removal of multimycotoxin because both the adsorbent and the catalyst can be designed, and the outcomes of this work also open a gate for the development of multifunctional matrix (i.e., for enzyme immobilization and reactant/inhibitor adsorption).

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.iecr.9b02094. AFB1 adsorption isotherm; procedures and results of MS analysis for AFB1 degradation products (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +010-62543301. E-mail: [email protected]. ORCID

Jianquan Luo: 0000-0002-9949-7779 Benkun Qi: 0000-0003-0158-3474 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial supports was supplied by the National Key Research and Development Plan of China (2017YFC1600906) and Beijing Natural Science Foundation (2192053).

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NOMENCLATURE AFB1 aflatoxin B1 HRP horseradish peroxidase SA/CS/MON sodium alginate/chitosan/montmorillonite AFs aflatoxins IARC International Agency for Research on Cancer Ca−Alg calcium alginate SA sodium alginate MON Montmorillonite CS−CaCl2 chitosan−calcium chloride BSA bovine serum albumin SEM scanning electron microscope EDX energy-dispersive X-ray spectroscopy FTIR Fourier transform infrared spectroscopy SD swelling degree HPLC high-performance liquid chromatography FLD fluorescence detector MS mass spectrometry Si silicon Al aluminum S sulfur

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