Co-immobilization of laccase and TEMPO onto amino-functionalized magnetic Fe3O4 nanoparticles and its application in acid fuchsin decolorization
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Laccase, a multicopper oxidase that catalyzes the oxidation of phenols, aromatic amines, and benzenethiols, has attracted much attention in applications of organic synthesis, bioremediation, and pulp/textile bleaching. However, free laccases cannot be recycled and are easily inactivated in diverse environmental conditions. Enzyme immobilization is a promising strategy to improve stability, resistance to extreme conditions, and reusability of laccase.
In this study, amino-functionalized magnetic Fe3O4 nanoparticles were synthesized for co-immobilization of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and laccase by glutaraldehyde cross-linking method. The magnetic nanoparticles were characterized with FTIR, XRD and VSM. Cyclic voltammetry was carried out to verify electrochemical behaviors of the co-immobilized laccase and TEMPO nanoparticles. When the co-immobilized laccase and TEMPO nanoparticles were used to decolorize acid fuchsin, the maximum decolorization rate of 77.41% was obtained with the ratio of TEMPO to laccase being 0.3 mM/g:120 U/g.
The co-immobilized nanoparticles retained above 50% residual activity after eight cycles of operation, which presented an approach to develop a co-immobilized laccase and mediator system for potential industrial application.
KeywordsAmino-functionalized magnetic Fe3O4 nanoparticles Co-immobilization Laccase TEMPO Decolorization
Fourier transfer infrared spectroscopy
vibrating sample magnetometer
laccase mediator system
Laccase (benzenediol:oxygen oxidoreductase, EC 188.8.131.52) is a multicopper oxidase and widely discovers from many plants, insects, and fungi (Mate and Alcalde 2017). It contains four copper atoms in its catalytic center and catalyzes the oxidation of phenols, polyphenols, polyamines, and benzenethiols by reducing molecular oxygen to water (Chao et al. 2017; Su et al. 2018; Mate and Alcalde 2015). In the past years, laccases have attracted much attention and been used in various applications, including organic synthesis, bioremediation, pulp bleaching, and biofuel production (Jeon and Chang 2013; Kudanga and Le Roes-Hill 2014). However, free laccases cannot be recycled and are easily inactivated in diverse environmental conditions, which limit their further use in industry.
Immobilization is a promising strategy to improve stability, resistance to extreme conditions, and reusability of laccase (Ba and Kumar 2017). So far, laccase has been successfully immobilized with several methods, including entrapment, encapsulation, adsorption, covalent binding, and self-immobilization (Ba et al. 2013; Fernández-Fernández et al. 2013). Although the stability of the immobilized laccase against temperature, organic solvents, pH, storage, and operation has been greatly improved, the activity recovery is not always satisfactory. In addition, the ability of laccase to catalyze recalcitrant compounds is limited for its low redox potential. This can be overcome by establishing laccase mediator system (LMS) (Jeon and Chang 2013; Mogharabi and Faramarzi 2014). 2,2′-Azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), 1-hydroxybenzo-triazole (HBT), and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) are usually used as mediators to transfer electrons from substrate to laccase. However, these mediators are usually expensive and difficult to recover from reaction mixture, which hampers the applications of LMS. Attempts have been made to immobilize mediators to recycle them. Machado et al. (2015) reported the immobilization of 4-hydroxy-TEMPO on mesoporous silica using 1,4-diisocyanatobutane as the linking agent. Tucker-Schwartz et al. (2010) employed iron oxide (Fe3O4) superparamagnetic nanoparticles to immobilize TEMPO with strong metal-oxide-chelating phosphonates and azide/alkyne “click” chemistry. The resultant TEMPO-coated nanoparticles, with good TEMPO loading, can be used to efficiently catalyze the oxidation of a wide range of alcohols.
In recent years, magnetic nanoparticles have attracted widespread attention because of their large surface areas, nontoxicity, magnetic properties, and biocompatibility. As a support material, magnetic nanoparticles can be rapidly separated from solution by the application of an external magnetic field rather than by centrifugation, and they have been deemed to be efficient carriers for enzyme immobilization (Ansari and Husain 2012; Can et al. 2009; Torres-Salas et al. 2011; Xin et al. 2010). Ren et al. have successfully immobilized lipase onto magnetic iron oxide nanoparticles via a biomimetic coating, which significantly improved its thermal and pH stability (Ren et al. 2011). Aminated magnetic mesoporous silica, (Fe3O4@MSS)-NH2, was synthesized to immobilize laccase using covalent cross-linking methods (Huang et al. 2014). 2,4-Dichlorophenol was degraded by the immobilized laccase with a removal efficiency of 88%, and 61.5% activity was remained after five cycles of operation. Zheng et al. (2012) prepared magnetic silica composite particles functionalized with 3-aminopropyltriethoxysilane (APTES) for laccase immobilization with the activity recovery of 83.9%.
In the present study, we reported a system that co-immobilizes laccase and mediator, allowing the simultaneous reuse of both laccase and the mediator. Magnetic nanoparticles were successfully prepared and modified with 3-amino-propyltriethoxysilane (APTES) to introduce abundant amine groups onto their surfaces. Then, laccase and 4-amino-TEMPO were efficiently co-immobilized onto the amino-functionalized Fe3O4 nanoparticles, using glutaraldehyde as the cross-linking agent. Finally, the decolorization of acid fuchsin, a triphenylmethane dye, by the co-immobilized laccase mediator system was conducted to evaluate the stability and efficiency for its potential future applications.
Laccase (EC 184.108.40.206 from Trametes versicolor, 13.6U/mg) and ABTS were purchased from Sigma-Aldrich Co. Ltd. (St. Louis, MO, USA). APTES, 4-amino-TEMPO, glutaraldehyde (25%, v/v, aqueous solution), Coomassie Brilliant Blue, bovine serum albumin, acid fuchsin and polyvinyl alcohol (PVA) with 1750 ± 50 of polymerization and 98% of degree of hydrolysis were from Aladdin Co. Ltd (Shanghai, China). All other chemicals were of analytical grade unless otherwise mentioned.
Preparation and characterization of magnetic Fe3O4 nanoparticles
Magnetic Fe3O4 nanoparticles were synthesized with the co-precipitation method and further modified with amino groups according to the method reported previously (Yamaura et al. 2004). First, 1.04 g of FeCl3·6H2O and 0.4 g of FeCl2·4H2O were dissolved in 10 mL deionized water, and 25 mL NaOH solution (3.0 M) was added to the mixture with vigorously stirring at 70 °C. After 30 min reaction, by applying an external magnetic field, the resultant Fe3O4 nanoparticles were collected and washed several times with deionized water until pH 7.0. The FTIR spectra of the nanoparticles were recorded on a Fourier transformed infrared spectroscopy (Thermo, USA). The XRD spectra of the nanoparticles were analyzed with D8 Advance XRD (Bruker, Switzerland). VSM (EV7, ADE Technologies) was used to record the magnetic susceptibility of the nanoparticles.
Co-immobilization of laccase and TEMPO
The amount of TEMPO attached onto the nanoparticles was measured spectrophotometrically, monitoring the changes in the 4-amino-TEMPO concentration before and after the reaction as the changes in the absorbance at a wavelength of 230 nm (A230) with a UV–Vis spectrophotometer (Thermo/GENESYS 10S, USA).
The modified glassy carbon (GC) electrodes were used as a working electrode with an Ag/AgCl as reference electrode and a Pt wire as a counter electrode, respectively. The glassy carbon electrodes were modified as follows: 5 mg of immobilized laccase and co-immobilized laccase and TEMPO was mixed with 100 μL of 0.3% PVA solution to produce PVA/Fe3O4-Laccase and PVA/Fe3O4-Laccase–TEMPO colloids, respectively. The surface of GC electrodes (5 mm in diameter) was polished thoroughly with Al2O3 (0.05 μm) by rinsing thoroughly with distilled water. Then, 10 μL PVA/Fe3O4-Laccase and PVA/Fe3O4-Laccase–TEMPO colloids solutions were dropped on the surface of pretreated electrodes and allowed to dry under ambient condition at 4 °C, respectively. After the modified electrodes were rinsed with distilled water twice or thrice, PVA/Fe3O4-Laccase and PVA/Fe3O4-Laccase–TEMPO modified GC electrodes were obtained. Experiments of cyclic voltammetry were run in a three-electrode system (PGSTAT302N, Metrohm, Switzerland) at a scan rate of 50 mV/s in pH 4.5 acetate butter solution containing 0.1 mM catechol.
Decolorization of acid fuchsin
Assay of enzyme activity
Results and discussion
Characterization of amino-functionalized Fe3O4 nanoparticles
Cyclic voltammetry of PVA/Fe3O4-Laccase GC and PVA/Fe3O4-Laccase–TEMPO modified GC electrodes
Effect of TEMPO on the decolorization of acid fuchsin by the immobilized laccase
Effect of the ratio of TEMPO to laccase on the co-immobilized laccase and TEMPO
Reusability of the co-immobilized laccase and TEMPO onto Fe3O4 nanoparticles
In summary, amino-functionalized magnetic Fe3O4 nanoparticles were prepared successfully by modifying magnetic nanoparticles to carry abundant amine groups on their surfaces. Laccase and 4-amino-TEMPO were co-immobilized onto the amino-functionalized magnetic Fe3O4 nanoparticles and used to decolorize acid fuchsin. The catalytic current of Fe3O4/PVA/Lac–TEMPO GC electrode is larger than Fe3O4/PVA/Lac GC electrode, which indicated that TEMPO was successfully immobilized onto carriers. When the ratio of TEMPO to laccase was 0.3 mM/g:120 U/g on the co-immobilized laccase and TEMPO nanoparticles, the maximum decolorization rate of acid fuchsin was 77.41%. Additionally, the co-immobilized nanoparticles retained above 50% residual activity after eight cycles of operation. This study offers a feasible approach to establish a co-immobilized laccase and mediator system, which could be useful in industrial applications.
HJ and PW designed the experiments, ZG and YY conducted most of the experiments, JZ analyzed the data, YX and XY wrote the manuscript, MJ, FC and HZ provided advices on the experimental design and language. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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The datasets supporting the conclusions of this article are included in the main manuscript.
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This research was financially supported by NSFC (21406114 and 20906048), the Natural Science Foundation of Jiangsu Higher Education Institutions of China (14KJB530002), the Science Foundation for Postdoctoral Research from Jiangsu Province of China (1401009A), the Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture, PCSIRT (IRT_14R28), and PAPD.
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- Xin B-J, Si S-F, Xing G-W (2010) Protease immobilization on γ-Fe2O3/Fe3O4 magnetic nanoparticles for the synthesis of oligopeptides in organic solvents. Chem Asian J 5:1389–1394Google Scholar
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