Article pubs.acs.org/Langmuir
Cite This: Langmuir XXXX, XXX, XXX−XXX
Protecting Enzymatic Activity via Zwitterionic Nanocapsulation for the Removal of Phenol Compound from Wastewater Guiqin Zheng,†,§ Shan Liu,†,§ Junqi Zha,† Peng Zhang,‡ Xuewei Xu,‡ Yantao Chen,*,† and Shaoyi Jiang*,‡ †
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Shenzhen Key Laboratory of Environmental Chemistry and Ecological Remediation, College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen, Guangdong 518060, China ‡ Department of Chemical Engineering, University of Washington, Seattle, Washington 98195, United States S Supporting Information *
ABSTRACT: Horseradish peroxidase (HRP) holds great potential in wastewater treatment. However, its instability in harsh environments remains a major issue. Various immobilization technologies were developed to retain enzyme stability at the cost of its effectiveness. We demonstrate that zwitterionic encapsulation of HRP retained both protein stability and activity to a large degree. In a water treatment study, encapsulating HRP into a zwitterionic nanogel resulted in a three-fold increase in the catalytic oxidation efficiency of phenol molecules. In addition, zwitterionic nanocapsules exhibited the best performance when compared with nanocapsules made from other hydrophilic polymers. These results indicated that zwitterionic HRP nanocapsules hold great potential in the decontamination of organic pollutants from wastewater.
1. INTRODUCTION As highly selective and efficient biocatalysts, enzymes have been extensively applied in chemical synthesis, pharmacology, cosmetics, and food processing.1 Since the initial work of Klibanov and colleagues2 in 1980, enzyme-mediated catalysis has become an increasingly important method to remove toxic compounds from industrial wastewater. For example, horseradish peroxidase (HRP; EC 1.11.1.1.7) is quite effective in oxidizing a broad spectrum of organic pollutants such as phenols, biphenols, dyes, and some environmental hormones.3,4 HRP, containing a heme prosthetic group as the active site, can catalyze the one-electron oxidation of aromatic substrates to form radicals.5 When dealing with the phenol compound, the generated free radicals polymerize with phenol monomers to form insoluble high-molecular-weight oligomers,2,6 and the latter could be easily removed by the subsequent filtration or sedimentation operations. Despite the advantages, the application of HRP in wastewater treatment is still limited by its inadequate enzymatic stability under the harsh reaction conditions. In addition to thermal unfolding, the enzyme active site is easily inhibited by the phenoxy radicals or the polymers generated during the oxidizing reaction.2 Various enzyme immobilization © XXXX American Chemical Society
and encapsulation methods have been developed to overcome these limitations.7−14 The immobilization/encapsulation with nanostructured materials brings immediate benefits to the enzymes, including enhanced stability, prevention of protein contamination, easy separation from the reaction mixture, and further modulation of the catalytic properties.4,15−17 To date, a variety of materials have been used to immobilize or encapsulate HRP, such as synthetic polymer, biopolymer, silica, and magnetic substances in the form of microspheres, nanoparticles, nanotubes, and porous structures.7−14 However, as a trade-off of the improved stability, the immobilization/ encapsulation may negatively affect enzyme activity by hindering the mass transfer of substrate and product, resulting in a slow catalytic rate and low effectiveness factor.16 As a result, industrial biotechnology is consistently seeking a Special Issue: Zwitterionic Interfaces: Concepts and Emerging Applications Received: June 14, 2018 Revised: July 19, 2018
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DOI: 10.1021/acs.langmuir.8b02001 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir
Figure 1. (A) Schematic showing of the synthesis of HRP zwitterionic nanocapsule. The free HRP (in cyan) contains one hemin group, in which porphyrin and iron atom are rendered as purple sticks and orange sphere, respectively. (B) AFM image of the HRP zwitterionic nanocapsule. (C) Size distributions and (D) zeta potentials of free HRP and HRP nanocapsule. The atomic force microscopy (AFM) measurements of samples were performed in a tapping mode with a Bruker MultiMode 8 apparatus. Dynamic light scattering (DLS) and zeta potential were measured using a Malvern Zetasizer Nano S90 apparatus. All bioassays were done using a BioTek Epoch 2 microplate spectrophotometer. 2.2. Enzyme Encapsulation by Zwitterionic Nanocapsule. A two-step protocol was adopted, which was first designed by Liu and coworkers.24 Enzyme was first acryloylated so as to prepare a macromonomer. Five milligrams of HRP was dissolved in 2 mL of PBS buffer (pH 7.4, 0.1 M). Then, 40 μL of NAS dissolved in DMSO with a concentration of 20 mg/mL was slowly added with a stirring speed of 800 rpm. The reaction was performed for 2 h at room temperature. After reaction completion, the acryloylated samples were concentrated and washed repeatedly with PBS 7.4 using 10 kDa molecular weight cutoff centrifugal filters. The number of reacted amino groups on HRP surface was determined by TNBS analysis.25 The HRP samples were first dissolved in carbonate-bicarbonate buffer (100 mM, pH 8.5). HRP solution (120 μL) with concentration from 15 to 500 mg/L was mixed with TNBS (50 μL, 5%) solution and then incubated at 37 °C for 2 h in the dark. Second, SDS solution (60 μL, 10%) and HCl solution (30 μL, 0.1 M) were added to terminate the reaction. The absorbance at 420 nm was then recorded on a microplate reader. Glycine solution was subjected to the same protocol to produce the calibration curve. Second, the acryloylated HRP was mixed with 100 mg CBAA and 20 mg MBA in PBS buffer (4.5 mL, pH 7.4). An in situ radical polymerization from the surface of the acryloylated HRP was initiated by APS (0.5 mL, 8 mg/mL) and TEMED (15 μL). After stirring for another 2 h at room temperature, the reaction mixture was concentrated and washed repeatedly with a 100 kDa molecular weight cutoff centrifugal filter. 2.3. Enzyme Activity Assays. A colorimetric method was employed to measure the catalytic activity of the free and encapsulated HRP. The assay solution was composed of TMB and hydrogen peroxide, both of which are substrates of HRP. Several
compromise between the catalytic performance and the environmental tolerance of the enzymes. In this study, we used the zwitterionic nanocapsulation technology to tackle this long-standing dilemma. HRP molecule was encapsulated into a zwitterionic poly(carboxybetaine) (PCB) hydrogel nanoparticle, and its potential application in wastewater treatment for the removal of phenol compound was explored. Because of the structural similarity between the PCB polymer and glycine betaine (a protein stabilizer), the conjugation of PCB has been shown to enhance protein stability without sacrificing its binding affinity.18−20 In addition, the superhydrophilic PCB polymer strongly resists nonspecific protein adsorptions.21,22 On the basis of these findings, we hypothesized that encapsulating HRP inside a zwitterionic nanogel could enhance its thermal stability and also avoid the inhibition from oxidized hydrophobic products. In the meantime, the porous gel network allows small organic pollutants and oxidized products to diffuse in and out, thus enabling it to act as a persistent catalyst support.
2. MATERIALS AND METHODS 2.1. Materials and Instruments. HRP (EC 1.11.1.7) was purchased from Sangon Biotech (Shanghai, China). N-Acryloxysuccinimide (NAS) and 2,4,6-trinitrobenzenesulfonic acid (TNBS) were obtained from TCI (Shanghai). Ammonium persulfate (APS), tetramethylethylenediamine (TEMED), phenol, 3,3′,5,5′-tetramethylbenzidine dihydrochloride (TMB), and N,N′-methylenebis(acrylamide) (MBA) were purchased from Sigma-Aldrich. Carboxybetaine acrylamide (CBAA) monomer was synthesized following a published method,23 the chemical structure of which is shown in Figure 1A. Other chemicals were of analytical grade and were used without further purification. B
DOI: 10.1021/acs.langmuir.8b02001 Langmuir XXXX, XXX, XXX−XXX
Article
Langmuir kinetic parameters of the Michaelis−Menten equation were interpreted using a Lineweaver−Burk plot, which are the Michaelis−Menten constant (Km), the turnover number (kcat), and the catalytic efficiency (kcat/Km). Experiments were performed in NaAc buffer solution (150 μL, 0.2 M, pH 5.5) with fixed concentration of H2O2 (1 mM) and varying concentration of TMB from 0.5 to 1.0 mM. Thermal stability tests of the free and encapsulated HRP were performed based on the same colorimetric method. The HRP samples were first incubated in a water bath at 55 °C with different time (from 0 to 180 min), then sequentially quenched in an ice bath before being subjected to activity assay. The final values were recorded as a percentage of each sample activity before heating. 2.4. Reaction and Detection of the Removal of Phenol Compound. Phenol removal experiments were conducted in a 30 °C water bath. Phenol (dissolved in distilled water) was mixed with PBS buffer solutions in the reactor with stirring at 100 rpm. When an adequate temperature of 30 °C had been reached, the enzyme suspension and the H2O2 solution were added sequentially. The phenol, H2O2, and HRP concentrations were set as 1 mM, 1 mM, and 10 μg/mL, respectively, according to the work of Cheng et al.26 Additional degradation tests with a large range of pH values (from 3 to 10) and substrate H2O2 concentrations (from 0.1 to 5 mM) were carried out to investigate the environmental tolerance of enzyme samples. The percentage of phenol compound was determined at different time intervals. The experiments were performed in triplicate, and data were expressed as mean values. The removal efficiency was estimated by measuring the concentration of residual phenol compound with a colorimetric method.27,28 The specimen including 200 μL of clear supernatant was sequentially mixed with 4-aminoantipyrine (25 μL, 21 mM) and potassium ferricyanide (25 μL, 83.4 mM). After 10 min, the color of the reaction mixture was developed completely, and the absorbance at 505 nm was detected.
thermal stress tests were performed by measuring the residual activity of the prepared samples against different elapsed times. As shown in Figure 2A, the relative activity of free HRP
Figure 2. Relative activities of free HRP and nanocapsule formulation incubated in a 55 °C water bath. (A) Protein concentration was fixed at 1 mg/L. (B) Elapsed time was fixed at 60 min for all samples.
3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of Nanocapsule. To prepare HRP nanocapsule, the enzyme was first chemically modified to introduce surface double bonds. The primary amine assay revealed that about three lysine residues were transformed into acryloyl groups, making the protein molecule a macromonomer. The protein macromonomer was then copolymerized with CBAA monomer and cross-linker (MBA) through in situ free radical polymerization reaction. Finally, the protein molecule was covered with a layer of zwitterionic PCB hydrogel, as shown in Figure 1A. The formation of HRP nanocapsule was verified by AFM imaging. As presented in Figure 1B, the dried nanoparticles were uniformly dispersed on mica with a mean diameter of
