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Supermagnetically tuned halloysite nanotubes functionalized with aminosilane for covalent laccase immobilization Avinash A. Kadam, Jiseon Jang, and Dae Sung Lee ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 18 Apr 2017 Downloaded from http://pubs.acs.org on April 19, 2017

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ACS Applied Materials & Interfaces

Supermagnetically tuned halloysite nanotubes functionalized with aminosilane for covalent laccase immobilization Avinash A. Kadam 1,† , Jiseon Jang,2,† and Dae Sung Lee2,*

1

Research Institute of Biotechnology and Medical Converged Science, Dongguk University, Biomedi Campus, Ilsandong-gu, Goyang-si, Gyeonggi-do, 10326, Republic of Korea 2

Department of Environmental Engineering, Kyungpook National University, 80 Daehak-ro, Buk-Gu, Daegu 41566, Republic of Korea

†

Avinash A. Kadam and Jiseon Jang contributed equally to this work.

*

To whom all correspondence should be addressed

Tel.: +82-53-950-7286, FAX: +82-53-950-6579, E-mail: [email protected]

KEYWORDS Nanobiocatalysis; Aminosilanization; Laccase; Magnetic halloysite nanotubes; Enzyme immobilization; Sulfamethoxazole 1

ACS Paragon Plus Environment


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ABSTRACT Halloysite nanotubes (HNTs) were tuned with supermagnetic Fe3O4 (M-HNTs) and functionalized with γ-aminopropyltriethoxysilane (APTES) (A-M-HNTs). Gluteraldehyde (GTA) was linked to A-M-HNTs (A-M-HNTs-GTA) and explored for the covalent laccase immobilization. The structural characterization of M-HNTs, A-M-HNTs, and A-M-HNTs-GTA immobilized laccase (A-M-HNTs-GTA-Lac) was determined by X-ray photoelectron spectroscopy, field-emission high resolution transmission electron microscopy, magnetic property measurement system, and thermogavimetric analyses. A-M-HNTs-GTA-Lac gave 90.20% activity recovery and loading capability of 84.26 mg/g, with highly improved temperature and storage stabilities. Repeated usage of A-M-HNTs-GTA-Lac revealed a remarkably consistent relative activity of 80.49% until the 9th cycle. The A-M-HNTs-GTA-Lac gave consistent redox mediated sulfamethoxazole (SMX) degradation up to the 8th cycle. In presence of guaicol, A-M-HNTs-GTA-Lac gave elevated SMX degradation compared with 2,2′azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) and syrinialdehyde. Therefore, the A-M-HNTs can serve as super-magnetic amino functionalized nanoreactors for biomacromolecule immobilization. The obtained A-M-HNTs-GTA-Lac is an environmentally friendly biocatalyst for effective degradation of micropollutants, such as SMX, and can be easily retrieved from an aqueous solution by a magnet after decontamination of pollutants in water and wastewater.

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1. INTRODUCTION Laccases (EC 1.10.3.2) belong to the multicopper oxidase protein family. Laccases monoelectronically oxidize various substrates to their corresponding radicals in the presence of molecular oxygen as an electron acceptor.1 This fact makes laccases highly fascinating for environmental and industrial applications.2 Many of the environmental pollutants are recalcitrant to oxidation by laccase3. However, laccases in the presence of redox mediators (small organic molecules that work as electron shuttles) can have mediated oxidative coupling reactions, where the mediator once oxidized to radicals by laccases is further reduced back to the original compound through the oxidation of a pollutant.4,5 This redox mediated reaction significantly increases their range of action towards the removal of various environmental pollutants from water and wastewater.3,5,6 Therefore, the broad substrate specificities of laccases make them excellent candidates to biocatalyze oxidation of environmental pollutants including micropollutants.3,4,7 The release of micropollutants into the environment in recent decades is one of the most concerning matters in developing water treatment protocols.8,9 Among these micropollutants, sulfamethoxazole (SMX) is one of the most extensively used pharmaceuticals and is typically known as an indicator of antibiotic pollution.10 Moreover, poor elimination of SMX from traditional municipal wastewater treatment plants imposes a potentially negative impact on aquatic organisms and so encourages the development of efficient treatment technologies.3,7,10 Amongst other available treatment technologies, the redox mediated bio-oxidation of SMX, catalyzed by laccase, is a very recent technology for SMX degradation.3,7 The commercial exploitation of laccase in such environmentally oriented applications has been predominantly relied on improved immobilization methods.11–13 These methods prominently simplify the 3



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burden of cost and enhance the stability of enzymes, and therefore are extensively pursued for selective, efficient, and environmentally friendly biocatalysis.12 Moreover, immobilized enzymes as a solid formulation, rather than liquid, allows their facile separation from the product and thus easily removing the protein contamination from the solution.14 Various supports such as organic, polymeric and inorganic materials have been investigated.13 Of them, inorganic materials outperforms organic materials in numerous characteristics, including antiswelling ability, better reusability, and microbial corrosion resistance.13 Despite these interesting features, the average internal pore size of inorganic materials is around 3-10 Å, which is the same as the size of small molecules. This limits their application for large and bulky biomacromolecules.13 Recently, inorganic materials such as halloysite nanotubes (HNTs, Al2Si2O5(OH)4·2H2O), that have hollow tubular structure with inner diameters of 5-20 nm, have been shown to be highly suitable for an extensive range of biomacromolecules,11,13,15. At moderate pH (3-10), HNTs’ outer surface gives negative zeta-potential behavior mainly because of the SiO2, but their inner surface is positively charged due to the Al2O3.11 In addition, HNTs as natural kaoline minerals are well known for biocompatibility, environmental friendliness, and low cost availability.16 These properties enable HNTs to have a wide scope of functionalizations for enzyme immobilization.13,15–19 The aminosilane or γ-aminopropyltriethoxysilane (APTES) functionalizations of HNTs for effective surface loading of precious metal nanoparticles such as Pt and Pd have been reported by several research groups.18–23 However, taking advantage of HNTs with aminosilanization for immobilization of laccase (pI 3.5-5), and other different biocatalysts is yet to be explored.11,16,24 Along with specific aminosilane functinilization of HNTs for improved loading, their excellent separation ability from solution is equally important for their practical application. Therefore, incorporation of the super-magnetic Fe3O4 property in HNTs provides a highly sophisticated 4


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technique to recycle and reuse the nanomaterials after completion of the process.25 To our knowledge, there are no reports available that have studied the incorporation of combined properties of super-magnetization and aminosilanization into the highly capable HNTs for excellent separation and effective enzyme loading, respectively. Therefore, the super-magnetic and aminosilane functionalized HNTs for enhanced loading of laccase in order to biocatalyze the degradation of SMX seem to be highly applicable nanoreactors in water treatment. In the current study, pristine HNTs were fabricated by incorporating super-magnetic (Fe3O4) property (M-HNTs), with further functionalization by grafting M-HNTs with the APTES (A-MHNTs). The glutaraldehyde (GTA) linked A-M-HNTs (A-M-HNTs-GTA) were explored for the covalent laccase immobilization. The detailed structural characterizations of M-HNTs, A-MHNTs, and A-M-HNTs-GTA immobilized laccase (A-M-HNTs-GTA-Lac) were carried out by fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), thermogavimetric analyses (TGA), field-emission high-resolution transmission electron microscopy (FE-HR TEM), fluorescent imaging under confocal laser scanning microscope (CLSM), and magnetic property measurement system (MPMS). The efficiently loaded laccase on A-M-HNTs-GTA (A-M-HNTs-GTA-Lac) were biochemically characterized for activity recovery, laccase loading, repeated reuse, and stability studies. Furthermore, A-M-HNTs-GTA-Lac was applied for continuous redox mediated biocatalytic degradation of micropollutant SMX in the presence of redox mediators such as guaicol (GUA), 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), and syringaldehyde (SA).

2. MATERIAL AND METHODS 2.1 Materials 5



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Laccase from Trametes versicolor, halloysite nanoclay, SMX, γ-APTES, fluorescein isothiocyanate (FITC), and ABTS were purchased from the Sigma Aldrich, USA. The SA and GUA were purchased from the Junsai, Japan. FeCl3·6H2O, FeCl2·4H2O, NH3·H2O, and gluteraldehyde (GTA) were obtained from Daejung chemicals, South Korea. 2.2 Synthesis 2.2.1 M-HNTs synthesis In order to incorporate magnetic (Fe3O4) property in HNTs, 0.5-1 g of HNTs was taken in 400 mL of distilled water and ultrasonicated (Powersonic 605) for 15 min at 25°C. Then this mixture was added with 2 g of FeCl3·6H2O and 1 g of FeCl2·4H2O at 60°C with constant stirring in the presence of the N2 gas. The NH3·H2O solution (22 mL, 28 vol.%) was added drop-wise into the mixture. The black precipitate of M-HNTs was aged at 60°C for 5 h. After the aging, MHNTs were separated by magnet, and washed thoroughly three times with distilled water, ethanol, and acetone, respectively. The finally obtained M-HNTs were dried at 60°C in an oven for 24 h. 2.2.2 Synthesis of A-M-HNTs and A-M-HNTs-GTA In a typical synthesis of A-M-HNTs, 5 mL of APTES was dissolved in 250 mL of anhydrous toluene, followed by the addition of 1.75 g of M-HNTs. Then the suspension was ultrasonicated (Powersonic 605) for 1 h at 20°C. The resultant mixture was taken into the reaction vessel, and the evacuation pretreatment was carried out. The suspension was then refluxed at 120°C for 24 h under constant stirring. In the refluxing system, the calcium chloride drying tube was attached in order to ensure a dry environment. Then, the obtained A-M-HNTs in the resultant mixture were separated magnetically. The separated A-M-HNTs were then extensively washed with fresh toluene in order to remove the excess aminosilane. The A-M-HNTs were then dried overnight at 6


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60°C, grounded in a mortar and pestle, and used for the subsequent experiments. The obtained A-M-HNTs nanocomposites were taken in the 2.5% of GTA solution. The resulting mixture was kept shaking at 150 rpm for 24 h. The A-M-HNTs-GTA was separated magnetically by the magnet, and washed thoroughly several times in order to remove unbounded GTA. The wellwashed A-M-HNTs-GTA was further explored in immobilization experiments. 2.3 Laccase immobilization and stability assessment The facile synthesized A-M-HNTs-GTA were used as a supporting material for the immobilization of laccase. In the typical immobilization process, A-M-HNTs-GTA were added into a 10 mL of laccase (1 mg/mL) solution in citrate phosphate buffer (pH 4.2). This mixture was kept on a shaking bed (150 rpm) at 25°C for 24 h. The resulting A-M-HNTs-GTA immobilized laccase (A-M-HNTs-GTA-Lac) was separated by a magnet, and washed with 20 mL of a citrate phosphate buffer (pH 4.2) solution for three times in order to remove the free laccase. Then, the wet A-M-HNTs-GTA-Lac was stored at 4°C for subsequent enzyme assays. The quantification of laccase loading on A-M-HNTs-GTA was determined by the Bradford method.26 The laccase activity was measured according to a previously reported method.13 The activities of free and A-M-HNTs-GTA-Lac were spectrophotometrically observed by generation of the ABTS radical after 5 min of gentle mixing in the citrate phosphate buffer (100 mM) at pH 4.2 containing ABTS (180 µM). The activity recovery (%) was calculated from the value of the activity of the initial laccase solution divided by the activity value of immobilized laccase obtained immediately after the immobilization procedure.13 The relative enzymatic activity was related to a percentage of the highest activity (100% represents the highest enzyme activity). In the typical resusabilty expiriments, the A-M-HNTs-GTA-Lac was magnetically removed after the first reaction cycle, and followed by the addition of the fresh reaction mixture for the next 7



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cycle. The kinetics of laccase were investigated by increasing ABTS concentrations (5-180 µM). The kinetic parameters of laccase such as Km and Vmax were calculated according to the Lineweaver–Burk double reciprocal models.7 In order to investigate the effect of pH, the free laccase and A-M-HNTs-GTA-Lac were kept at different pH 2-8 (50 mM, citrate-phosphate buffer pH 3-7, Tris-HCl buffer pH 8) for 1 h incubation at 30°C, and followed by measuring the residual enzyme activity. Temperature stability was studied by measuring the residual activity at 60°C in the citrate phosphate buffer (100 mM, pH 4.2) over time. The storage stability of free and immobilized laccase was analyzed by storing them at 4°C in citrate phosphate buffer (100 mM, pH 4.2) for 30 d with intermittent measurement of residual activity. 2.4 Fluorescent labeled M-HNTs, A-M-HNTs, and A-M-HNTs-GTA-Lac The fluorescent labeled M-HNTs, A-M-HNTs and A-M-HNTs-GTA-Lac were prepared according to the procedure reported by Dai et al28. The respective nanomaterials (50 mg) were taken in 1 mL of phosphate buffer solution (100 mM, pH 7.0). This mixture was mixed with 1.5 mL of 0.5 mM carbonate buffer solution (pH 9.0) under vigorous stirring. Further, this mixture was added with 0.3 mL of freshly prepared FITC solution (1 mg of FITC in 1 mL of 0.5 mM carbonate buffer solution as solvent, pH 9.0) and stirred for 2 h at room temperature. After that, fluorescently labeled M-HNTs, A-M-HNTs, and A-M-HNTs-GTA-Lac were separated by an external magnet and washed with 0.5 mM carbonate buffer solution to remove unbound FITC. The obtained samples were analyzed by a confocal laser scanning microscope (CLSM) under the excitation and emission wavelengths of 488 and 535 nm, respectively. 2.5 Redox mediated degradation of SMX by A-M-HNTs-GTA-Lac Redox mediated degradation of SMX was carried out by A-M-HNTs-GTA-Lac. In a typical reaction mixture, 0.5 ml of SMX (25 ppm) and 0.2 ml of A-M-HNTs-GTA-Lac in a citrate 8


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phosphate buffer of pH 6 were taken in the presence of different redox mediators such as guaicol (500 µM), SA(500 µM), and ABTS (500 µM), respectively. These reaction mixtures were incubated at 25oC in a shaking incubator at 120 rpm for 24 h. The degradation of SMX was monitored by high performance liquid chromatography (HPLC, Shimadzu LC20AD). The HPLC instrument was equipped with a Zorbax Eclipse XDB-C18 column (150 mm × 4.6 mm, 5 m) and a UV/vis detector (SPD-20A) was used in isocratic mode in order to analyze the SMX degradation. A mobile phase containing methanol/water (with 1% acetic acid) mixture (60:40, v/v) was used with a flow rate of 0.6 mL min−1. The detection wavelength was set at 286 nm. For the repeated SMX degradation experiment, immobilized laccase at the end of each cycle was removed by the magnet, washed with the citrate phosphate buffer of pH 6 and added to a new reaction mixture for the next cycle. 2.6 Structural characterizations A variety of oxygenated functional groups of M-HNTs, A-M-HNTs, and A-M-HNTs-GTALac were determined by FT-IR/NIR spectroscopy (Spectrum 100, PerkinElmer, USA). The structural morphology was analyzed by field emission scanning electron microscopy (FE-SEM, Hitachi S-4800) and FE-HR TEM (FEI, Titan G2 ChemiSTEM Cs Probe). XPS was used to analyze the surface elemental composition by using a ULVAC-PHI Quantera SXM. TGA of the samples was done from room temperature to 800°C at a heating rate of 10°C min-1 under a nitrogen atmosphere using a thermal analyzer system (TA Instrument, Q600). XRD analysis was performed using an X-ray diffractometer (Rigaku, D/Max-2500) with Cu Kα radiation (λ = 1.5406 Å) over a scanning range of 10° to 80° (2θ). FITC coated samples were observed under CLSM (LSM700) at the excitation and emission wavelengths of 488 and 535 nm, respectively.

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The supermagnetic properties of the samples were analyzed by a SQUID-VSM QM02 magnetometer at room temperature with an applied field between −15,000 and 15,000 Oe.

3. RESULTS AND DISCUSSION 3.1 Synthesis of A-M-HNTs In the most recent reports, the pristine and functionalized HNTs were described as striking materials for enzyme immobilization by several research groups.11,13,16,29 However, although the pristine and functionalized HNTs resulted in “templates and nanoreactors” for enzyme loading, their separation from aqueous solution have been done conventionally by filtration and centrifugation.11,13,29,30 This conventional seperation technique leads to an increase in total cost for the removal of enzyme loaded HNTs from the used solution. However, the incorporation of supermagnetism in supporting materials can solve problems such as enzyme recycling and substrate and product separation.31–33 Therefore, there is an ample space for the development of highly applicable HNTs with excellent supermagnetism for enzymatic reactions. Moreover, in order to maintain the most stable enzymes and their attachment to the supporting materials, the formation of covalent bonds is essential.32–34 For covalent loading of laccase on HNTs, their surfaces were functionalized with aminosilane APTES and subsequently the bifunctional crosslinking agent (GTA) was used, as it links functional groups (−NH2) from the supports and enzymes.33,35,36 Fig. 1 illustrates the schematic description of a typical strategy for the stepwise tuning and functionalization of pristine HNTs, designed for the covalent loading of laccase. In the first step, the pristine HNTs were tuned with super-magnetic Fe3O4 nanoparticles. In second step, M-HNTs were functionalized by the aminosilane APTES. Further, in the third and fourth

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step, GTA binding was carried out in order to covalently immobilize enzyme laccase on A-MHNTs-GTA.

Figure 1. The schematic description of A-M-HNTs synthesis and subsequent covalent immobilization of laccase. 3.2 Structural characterization The installation of supermagnetic Fe3O4 on HNTs and further chemical functionalization by APTES were confirmed by FT-IR analysis. Fig. 2 shows FT-IR spectrums of M-HNTs (green curve), A-M-HNTs (blue curve), and A-M-HNTs-GTA-Lac (pink curve). As can be seen from Fig. 2, the peaks of HNTs at 3695, 3620, 1028, and 910 cm-1 were assigned to the O-H stretching of hydroxyl groups on the inner surface of HNTs, the O−H stretching vibration of the inner 11



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Al−OH groups (between the interface of the Si−O tetrahedron and the Al−O octahedron), O−H deformation of inner hydroxyl groups, and in-plane stretching vibration of the Si−O network (Si−O−Si and O−Si−O), respectively18,23,37.

Figure 2. FT-IR spectrum of M-HNTs, A-M-HNTs, and A-M-HNTs-GTA-Lac. The absorption band observed at 536 cm−1 denotes the bending vibration of Fe–O bond of Fe3O4 and confirms a successful loading of the Fe3O4 on the pristine HNTs.38 All of these characteristic peaks of M-HNTs remained in both A-M-HNTs and A-M-HNTs-GTA-Lac, indicating that the basic backbone structure of M-HNTs was constant after functionalization and laccase loading. After the functionalization of M-HNTs by APTES (A-M-HNTs), new characteristic peaks were exhibited at 3368, 2925, 2854, and 1245 cm−1 which were designated to the asymmetric stretching vibration of N−H2, asymmetric stretching vibration of C−H2, symmetric stretching 12


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vibration of C−H2, and vibration of the C-N bond, respectively20,23,37. Thus, the absorption peaks in A-M-HNTs confirmed the successful grafting of aminosilane APTES on the M-HNTs.18–20,23 These characteristic peaks in A-M-HNTs were also consistent in A-M-HNTs-GTA-Lac (Fig. 2). The covalent attachment of the enzyme on the supporting material was shown by characteristic peaks for amide I and II in the respective FT-IR spectrum.39,40 The detailed information of the secondary structure of the enzyme were presented by the shapes of the amide I and II infrared absorbance bands.29 Herein, the FT-IR spectrum of A-M-HNTs-GTA-Lac represented the absorption peaks at 1649 and 1549 cm−1, which were assigned to the amide I C=O stretching modes and a characteristic amide II band, respectively.39 As a result, these peaks gave strong confirmation regarding the covalent immobilization of laccase on the A-M-HNTs. Therefore, FT-IR spectra gave detailed information about presence of magnetite on HNTs, APTES functionalization of M-HNTs, and covalent laccase loading on A-M-HNTs. The crystallinity and purity of the chemically modified HNTs were confirmed by XRD analysis (Fig. S1). The basal space reflections indicated a sharp peak at (2θ =12.09) in XRD spectra of M-HNTs, A-M-HNTs, and A-M-HNTs-GTA-Lac samples, corroborating to a (001) basal spacing of 0.73 nm (JCPDS Card No. 29-1487), which confirmed the nanotubular multiwall structure of HNT.29,41,42 These results depict that the HNTs served as a nanotubular backbone in M-HNTs, A-M-HNTs, and A-M-HNTs-GTA-Lac. The characteristic XRD peaks for Fe3O4 (2θ = 30.21, 35.5, 43.25, 57.1 and 62.7), marked by their indices (220), (311), (400), (422), and (511) and further coordinated with the database of magnetite in the JCPDSInternational Center (JCPDS card: 19-0629)38, were almost the same in M-HNTs, A-M-HNTs and A-M-HNTs-GTA-Lac. Therefore, the magnetic Fe3O4 nanoparticles were successfully installed on the surface of HNTs. Moreover, it also could be seen that the XRD pattern of M13



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HNTs, A-M-HNTs, and A-M-HNTs-GTA-Lac was similar, indicating they possessed the same cylinder-wall structure and inter-planar spacing. Similar observation of XRD data were corroborated by the loading of APTES on HNTs.20 Hence, the observed XRD data implied that there was no phase change in HNTs after their magnetization, functionalization with APTES and subsequent loading of laccase. Further, more detailed investigation of the respective chemical composition of modified HNTs was completed using XPS analysis. Fig. 3 (a) represents the XPS spectrum of M-HNTs, A-M-HNTs, and A-M-HNTs-GTA-Lac, and their relative atomic concentrations (%) are described in Table 1. The Fe 2p spectrum gave two bands at 713.96 and 727.03 eV, which corresponding to the formation of Fe3O4 (Fig.S2).43 The elaborated XPS spectra of N 1s, represented in Fig 3 (b), shows more sharp N 1s peak in A-M-HNTs and A-M-HNTs-GTA-Lac, compared with the M-HNTs. In addition, Table 1 reveals a significant increase in nitrogen concentration (%) in A-M-HNTs and A-M-HNTs-GTA-Lac compared with the M-HNTs, validating efficient loading of the amino group containing APTES and laccase on the respective supporting material. Moreover, atomic carbon (C 1s) concentration significantly increased in AM-HNTs and A-M-HNTs-GTA-Lac, which may be due to enhanced loading of APTES containing the carbon side chain, and the laccase exhibiting the carbon throughout its structure, respectively. Elements such as Si and Al were detected in all samples. The Si/Al concentration ratio on the surface of modified HNTs was observed to be higher than that of M-HNTs (Table 1). These values confirmed the presence of silicon from APTES on the surface of M-HNTs. Similar enhancement in Si/Al ratio after the APTES modification of HNTs has been detected earlier by Zhang et al23. Therefore, the obtained results strongly corroborate the successful grafting of APTES on M-HNTs surface. 14


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The thermal degradation patterns of the M-HNTs, A-M-HNTs, and A-M-HNTs-GTA-La were observed by TGA analysis (Fig. S3). The M-HNTs, A-M-HNTs, and A-M-HNTs-GTA-Lac in the temperature range between 25 to 796°C show weight losses of 13.11, 13.99, and 21.22%, respectively. Hence, A-M-HNTs and A-M-HNTs-GTA-Lac display 0.88%

Figure 3. (a) XPS analysis of M-HNTs, A-M-HNTs, and A-M-HNTs-GTA-Lac and (b) their respective representative N 1s peak.

and 8.11% more weight loss than the control backbone M-HNTs. The increased weight loss in the A-M-HNTs corroborates to the thermal loss of loaded APTES on M-HNTs, indicating that 15



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the loaded amount of the APTES over the M-HNTs were approximately 0.88% of the weight.13 Moreover, A-M-HNTs-GTA-Lac gives 8.11% and 7.23% more weight loss than M-HNTs and A-M-HNTs, respectively. These results gave a very strong corroboration regarding the successful loading of laccase on A-M-HNTs. Table 1. Atomic concentration (%) of elements of support materials M-HNTs, A-M-HNTs, and A-M-HNTs-GTA-L. Atomic concentration (%)

Concentration ratio

Sample

C1s

N1s

O 1s

Si 2p

Al 2p

Si/Al

M-HNTs

50.8

0.2

42.0

3.8

3.3

1.1

A-M-HNTs

54.2

3.5

34.9

4.6

2.8

1.6

A-M-HNTs-GTA-Lac

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The structural morphology of pristine HNTs and A-M-HNTs was investigated by FE-HR-TEM imaging. Fig. 4 (a-b) displays the hollow tubular cylindrical morphology and open ends in both HNTs and A-M-HNT. Fig. 4 (b) shows the FE-HR-TEM image of single representative HNT with the dimensions of 633 nm of height and a diameter of 131 nm, respectively. Furthermore, Fig. 4 (a-b) reveals that the characteristic cylindrical tube morphology of HNTs has been retained intact in the modified A-M-HNTs. This observation was in accordance with the XRD analysis (Fig. S1). The effective distribution of magnetic Fe3O4 nanoparticles on the HNTs surface is noticeably observable in Fig. 4 (b-c). The crystalline information of magnetic Fe3O4 nanoparticles anchored on HNTs is shown in Fig. 4 (b[i]). The selected-area electron diffraction (SAED) analysis of anchored Fe3O4 on HNTs corroborates their crystalline nature. Crystal planes of Fe3O4 (111), (220), (400), (311), (422), (511), and (422) in Fig. 4 (b[i]) are consistent with the 16


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crystal planes obtained in the XRD pattern (Fig. S1). The particle size of homogeneously distributed Fe3O4 on the HNTs is in the range of 8-10 nm (Fig. 4 (c)). Similar TEM images of Fe3O4 nanoparticles on the HNTs were observed by a previous report.25,38 In addition to the surface distribution, effective loading was also observed on to the ends of the hollow tubular HNTs (represented by the red arrows in Fig. 4 [b]), giving a visual insight that effective loading also occurred throughout the hollow lumen of the HNTs. Therefore, from all of these observations from the FE-HR-TEM analysis, it is concluded that HNTs provide a background support for APTES functionalization and serve as nanoreactors for laccase catalysis.

Figure 4. Typical FE-HR-TEM images of (a) pristine HNTs, (b) representative A-M-HNT with marked dimensions and involving the SAED pattern of anchored Fe3O4 (i), and (c) A-M-HNTs with surface loading. Similarly Fig.S4 represents the FE-SEM of image of A-M-HNTs, which revealed the number of halloysite nanotubes of various sizes were decorated by Fe3O4 nanoparticles. Although modified HNTs in TEM images might suggest respective loading on HNTs, clear visual imaging is required to confirm successful loading. FITC dye binds to the amino functional group and has been widely used for confirmation of amino-modified nanoparticles and successful enzyme 17



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loading.28,44 Therefore, aminosilane grafting and laccase immobilization on modified HNTs was simultaneously studied through CLSM by FITC coating (Fig. 5). No fluorescence was observed in the CLSM image of M-HNTs (Fig. 5a). However, a clear green fluorescence can be observed in the images of A-M-HNTs and A-M-HNTs-GTA-Lac, which visually confirms successful aminosilane APTES grafting and laccase loading. Moreover, short nanotubes of modified HNTs (marked by red circles in Fig. 5) with an average length of approximately 0.5-1 µm can be observed in Fig. 5 (b & c).

Figure 5. CLSM images of FITC lebelled (a) M-NHTs, (b) A-M-HNTs, and (c) A-M-HNTsGTA-Lac. The magnetic properties of A-M-HNTs and A-M-HNTs-GTA-Lac were investigated by MPMS analysis. The A-M-HNTs and A-M-HNTs-GTA-Lac gave the magnetization saturation values of 41.10 and 37.03 emu/g, respectively (Fig. 6). This magnetic saturation values confirmed the super-magnetic nature of the A-M-HNTs and A-M-HNTs-GTA-Lac. Additionally, the coercivity and remanence values of the curve were observed to be zero, also representing their super-magnetic nature. However, the A-M-HNTs-GTA-Lac exhibited 4.07 emu/g less magnetization saturation than the A-M-HNTs. The magnetization saturation value of a material 18


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is mainly affected by its crystallinity and an increase in size due to the loading of bulky material.38 Therefore, it proves that the magnetic saturation value decreased in A-M-HNTsGTA-Lac with the covalent loading of laccase on A-M-HNTs. However, the super-magnetism of both the samples was excellent for easy and quick separation from the suspension (Fig. 6).

Figure 6. MPMS analysis of A-M-HNTs and A-M-HNTs-GTA-Lac. Additionally, the magnetic saturation capacity of A-M-HNTs in this study is amongst the highest magnetism for all of the reported magnetic HNTs (supporting information, Table S1). Furthermore, despite the importance of modified HNT as a nanoreactor system for enzyme biocatalysis, to our knowledge, there are no previous reports available for modified HNTs for effective enzymatic biocatalysis with excellent magnetic separation (Table S1). Therefore, this study demonstrates the excellent magnetic separation of modified HNTs for enzyme biocatalysis. 19



In addition, the detailed structural characterization made by different analytical techniques such as FT-IR, XPS, TGA, FE-HR-TEM, FITC coating, and MPMS clearly confirmed the covalent immobilization of laccase on A-M-HNTs.

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0 HNTs

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Immobilization support material Figure 7. Laccase loading and activity recovery (%) on HNTs, M-HNTs, and A-M-HNTs-GTA. 3.3 Properties of A-M-HNTs-GTA-Lac Biochemical behavior and stability assays after the successful immobilization process were investigated. A-M-HNTs-GTA-Lac gave 90.20% of activity recovery, while the laccase immobilized on pristine HNTs and M-HNTs gave 15.78% and 30.51% of activity recovery, respectively (Fig. 7). At the same time, pristine HNTs, M-HNTs, and A-M-HNTs-GTA-Lac showed 7.9, 13.0, and 84.26 mg/g of the laccase loading over the respective supporting materials (Fig. 7). Therefore, subsequent and stepwise modifications such as incorporation of 20


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supermagnetism and aminosilane functionalization in HNTs might lead to an enhanced laccase activity recovery and protein loading, respectively. However, when the laccase concentration was more than 1 mg/mL, a decrease in the activity recovery of the A-M-HNTs-GTA-Lac was observed (Fig. 8 [a]). Similar observations were also obtained by some previous supporting materials.13,45 This might be due to the fact that excessive loading of laccase would easily lead to the agglomeration or crowding of enzyme molecules onto the surface of supports. Hence, the optimum laccase concentration was taken as 1 mg/mL for further studies. Meanwhile, at the initial laccase concentration of 1 mg/mL, the enzyme loading on the modified HNTs with chitosan and polydopamine were 15 and 25 mg/g, respectively.13,45 However, in this study, the A-M-HNTs-GTA-Lac gave five and three times higher loading capacities, respectively. This observation demonstrates very high enzyme loading capacity of the A-M-HNTs-GTA. The Km and Vmax values of free laccase and A-M-HNTs-GTA-Lac were determined based on the Lineweaver–Burk plot (Fig. S5, Table S2). The free laccase and A-M-HNTs-GTA-Lac gave the Km values of 80 and 90 µM, respectively. The increase in Km denotes a decrease in affinity of enzyme towards the substrate. This observation was similar with the previous report, in which a decrease in affinity were observed after the covalent immobilization.7 The mass transport constraint and proteins structural changes after covalent binding might contribute to the decreased in affinity.7 Stabilities of the biocatalyst are important and a prerequisite when considering its commercial industrial applications. A-M-HNTs-GTA-Lac gave good stability in a broad pH range (2-8) compared to the free laccase (Fig. S6). As the pH decreased from 5 to 2, the free laccase and A-M-HNTs-GTA-Lac retained 22 and 51% of their relative activity, respectively (Fig. S6). Similarly, as the pH increased from 5 to 8, A-M-HNTs-GTA-Lac held 46% of the relative activity, while free laccase lost 92% of its relative activity (Fig. S6). This 21



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result corroborates the stability of the A-M-HNTs-GTA-Lac in a broad range of pH. Similar results were observed by immobilized laccase on the porous silica beads.46 In thermal stability studies (Fig. 8[b]) at a temperature of 60°C, the free laccase lost around 40% of its initial activity in 360 min. While the A-M-HNTs-GTA-Lac retained the 91% of its initial activity, and hence represented very high thermal stability. Similarly, the storage time stability was represented in Fig. 8c. After 30 d of incubation at 4°C, the free laccase lost its half of its initial activity, but the A-M-HNTs-GTA-Lac retained 87% of its initial activity. The improved storage stability of A-MHNTs-GTA-Lac might be the result of covalent cross linkages between the enzyme molecules through GTA, which prevented enzyme leaching, enzyme denaturation, and autodigestion. Furthermore, the backbone HNTs served as nanoreactors and may help maintain the active conformation of the enzyme at higher temperatures. These results confirm the high thermal and storage stabilities of A-M-HNTs-GTA-Lac. The improved thermal and storage stabilities of AM-HNTs-GTA-Lac would extend the application of the immobilized laccase. Along with high thermal and storage stabilities, excellent reusability is also required for the desired practical application of immobilized enzymes in industry. Fig. 8 (d) shows the reusability capacity of the A-M-HNTs-GTA-Lac. The 14% loss of the initial laccase activity was observed up to the successive 8th cycle. However, the activity from the 9th cycle continued in a state of slowdown for the next four cycles. The activity loss during the cycles of repeated use could be due to enzyme leakage during continuous washing and enzyme deactivation during repeated use. However, even after the completion of 12 repeated uses, the A-M-HNTs-GTA-Lac possessed more than 67% of its initial activity. However, HNTs modified with the chitosan represented a 50% loss of the initial activity only after the fifth cycle.47 Similarly, polydopamine-modified HNTs showed a 70% retention of the initial activity after the 10th cycle. 22


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Figure 8. (a) Laccase loading and activity recovery (%) on A-M-HNTs-GTA with increase in initial laccase concentration, (b) thermal stability studies of A-M-HNTs-GTA-Lac at 60°C up to 360 min, (c) storage stability of A-M-HNTs-GTA-Lac at 4°C up to 30 d, (d) reusability of the AM-HNTs-GTA-Lac in continuous cycles. 13 However, A-M-HNTs-GTA-Lac exhibited a 78% retention of the initial activity after the 10th cycle of reuse. This shows the high potential of A-M-HNTs-GTA-Lac for practical applications of laccase. 3.4. Removal of SMX

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Laccases can catalyze the oxidation of different micropollutants such as bisphenol A, triclosan, diclofenac, mafenamic acid, isoproturon, fluoroquinolones, tetracyclines, and sulfonamide antibiotics.3,7,48 In this study, SMX was selected as the target contaminant for A-MHNTs-GTA-Lac. Laccase alone without the redox mediators was unable to degrade SMX.3,7 The chemical structures of SMX, GUA, ABTS and SA are given in Fig. S7. The removal of SMX by free laccase in the presence of redox mediators such as ABTS, SA, and GUA were 32.51%, 36.3%, and 45.76%, respectively (Fig. 9). However, A-M-HNTs-GTA-Lac in the presence of mediators ABTS, SA, and GUA exhibited 43.06%, 54.82%, and 68.88% of the SMX removal, respectively (Fig. 9). Therefore, the A-M-HNTs-GTA-Lac in the presence of mediators showed a higher removal of SMX compared to the free laccase. Similar observation were also reported by an earlier study.7 This may be due to the change in the affinity between laccase and mediator, which has a direct impact on the catalytic rate.7 Hence, the A-M-HNTs-GTA-Lac might change the affinity towards the mediators and result in higher catalytic rates. Meanwhile, among the three different mediators studied, GUA was the most effective laccase mediator for an increased removal of SMX (Fig. 9). Indeed, GUA was effectively used as an indicative benchmark substrate in order to engineer laccase towards more efficient “green” applications.49

24


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Figure 9. SMX removal by free laccase (FL) and immobilized laccase (IL) in the presence of redox mediator ABTS, SA, and GUA (Reaction conditions: pH 6, Temperature 25oC in a shaking incubator at 120 rpm for 24 h). Moreover, GUA has been reported to be a more beneficial substrate than ABTS and DMP (2,6dimethoxyphenol) in directing the protein engineering of laccases toward realistic bioremediation.49 Furthermore, successive GUA mediated degradation of SMX using A-MHNTs-GTA-Lac has been investigated in nine successive degradation-regeneration cycles (Fig. S8). A-M-HNTs-GTA-Lac gave a 60% removal of SMX up to the 7th cycle, and subsequently decreased to 41.65%. Hence, the repeated SMX degradation capacity of the A-M-HNTs-GTALac confirms their potential application in the field of water treatment. By taking into account 25



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thermal and storage stabilities, excellent reusability, and successive redox mediated SMX degradation, the A-M-HNTs-GTA-Lac is highly appropriate for practical water treatment applications.

4. CONCLUSIONS This study demonstrated the stepwise modification of HNTs by super-magnetic tuning with Fe3O4, followed by aminosilane functionalization. Successful synthesis of modified HNTs was confirmed by structural characterizations using FT-IR, XRD, XPS, TGA, FE-HR-TEM, and MPMS. The synthesized A-M-HNTs-GTA-Lac gave improved laccase loading and activity recovery compared with pristine HNTs. A-M-HNTs-GTA-Lac exhibited enhanced thermal and storage stabilities with excellent reusability. The A-M-HNTs-GTA-Lac was used in successive degradation-regeneration cycles for redox mediated degradation of SMX, where GUA was more effective mediator than ABTS and SA. Therefore, this study concludes that robust, supermagnetic and aminosilane functionalized A-M-HNTs can serve as a nanoreactor for biomacromolecule immobilization. In addition, as the obtained A-M-HNTs-GTA-Lac is an environmentally friendly biocatalyst and can be easily retrieved from an aqueous solution by a magnet after decontamination of target pollutants, it has extensive applicability for the removal of micropollutants from water and wastewater.

Supporting Information XRD patterns of M-HNTs, A-M-HNTs and A-M-HNTs-GTA-Lac, Fe 2p XPS spectrum of A-MHNTs, TGA analysis of M-HNTs, A-M-HNTs and A-M-HNTs-GTA-Lac, FE-SEM image of A26


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M-HNTs, detailed review of magnetically modified HNTs for different functions, Lineweaver– Burk double reciprocal plots for determination of the Km of freee laccase and A-M-HNTs-GTALac, Kinetic constants of free laccase and A-M-HNTs-GTA-Lac, the effect of pH on stability of free laccase and A-M-HNTs-GTA-Lac, chemical structures of SMX, GUA, ABTS and SA, repeatative SMX removal by A-M-HNTs-GTA-Lac in presence of GUA.

ACKNOWLEDGEMENTs This work was supported by the Human Resource Training Program for Regional Innovation and Creativity through the Ministry of Education (ME) and National Research Foundation (NRF) of Korea (NRF-2014H1C1A1066929). This study was also supported by grants (NRF2016R1A2B4010431 and NRF-2009-0093819) through the ME and NRF of Korea. This research was also supported by an NRF grant from the Korean government (MSIP) (NRF2015M2A7A1000194).

REFERENCES (1)

Jiang, J.; Ye, W.; Liu, L.; Wang, Z.; Fan, Y.; Saito, T.; Isogai, A. Cellulose Nanofibers Prepared Using the TEMPO/Laccase/O 2 System. Biomacromolecules 2016, 18, 288-294.

(2)

Camarero, S.; Cañas, A. I.; Nousiainen, P.; Record, E.; Lomascolo, A.; Martínez, M. J.; Martínez, Á. T. P-Hydroxycinnamic Acids As Natural Mediators for Laccase Oxidation of Recalcitrant Compounds. Environ. Sci. Technol. 2008, 42, 6703–6709.

(3)

Margot, J.; Copin, P. J.; von Gunten, U.; Barry, D. A.; Holliger, C. Sulfamethoxazole and Isoproturon Degradation and Detoxification by a Laccase-Mediator System: Influence of 27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 34

Treatment Conditions and Mechanistic Aspects. Biochem. Eng. J. 2015, 103, 47–59. (4)

Telke, A. A.; Kalyani, D. C.; Jadhav, U. U.; Parshetti, G. K.; Govindwar, S. P. Purification and Characterization of an Extracellular Laccase from a Pseudomonas Sp. LBC1 and Its Application for the Removal of Bisphenol A. J. Mol. Catal. B: Enzym. 2009, 61, 252–260.

(5)

Husain, M.; Husain, Q. Applications of Redox Mediators in the Treatment of Organic Pollutants by Using Oxidoreductive Enzymes: A Review; Crit. Rev. Environ. Sci. Technol. 2007, 38, 1-42.

(6)

Feng, Y.; Colosi, L. M.; Gao, S.; Huang, Q.; Mao, L. Transformation and Removal of Tetrabromobisphenol A from Water in the Presence of Natural Organic Matter via Laccase-catalyzed Reactions: Reaction Rates, Products, and Pathways. Environ. Sci. Technol. 2013, 47, 1001-1008.

(7)

Shi, L.; Ma, F.; Han, Y.; Zhang, X.; Yu, H. Removal of Sulfonamide Antibiotics by Oriented Immobilized Laccase on Fe3O4 Nanoparticles with Natural Mediators. J. Hazard. Mater. 2014, 279, 203–211.

(8)

Zearley, T. L.; Summers, R. S. Removal of Trace Organic Micropollutants by Drinking Water Biological Filters. Environ. Sci. Technol. 2012, 46, 9412–9419.

(9)

Cao, X.; Luo, J.; Woodley, J. M.; Wan, Y. Bioinspired Multifunctional Membrane for Aquatic Micropollutants Removal. ACS Appl. Mater. Interfaces 2016, 8, 30511–30522.

(10)

Jiang, B.; Li, A.; Cui, D.; Cai, R.; Ma, F.; Wang, Y. Biodegradation and Metabolic Pathway of Sulfamethoxazole by Pseudomonas Psychrophila HA-4, a Newly Isolated Cold-Adapted Sulfamethoxazole-Degrading Bacterium. Appl. Microbiol. Biotechnol. 2014, 98, 4671–4681. 28


Page 29 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(11)

Tully, J.; Yendluri, R.; Lvov, Y. Halloysite Clay Nanotubes for Enzyme Immobilization. Biomacromolecules 2016, 17, 615-621.

(12)

Tran, D. N.; Balkus, K. J. Perspective of Recent Progress in Immobilization of Enzymes. ACS Catal. 2011, 1, 956–968.

(13)

Chao, C.; Liu, J.; Wang, J.; Zhang, Y.; Zhang, B.; Zhang, Y.; Xiang, X.; Chen, R. Surface Modification of Halloysite Nanotubes with Dopamine for Enzyme Immobilization. ACS Appl. Mater. Interfaces 2013, 5, 10559–10564.

(14)

Sheldon, R. A.; Van Pelt, S. Enzyme Immobilisation in Biocatalysis: Why, What and How. Chem. Soc. Rev. 2013, 42, 6223–6235.

(15)

Levis, S. R.; Deasy, P. B. Characterisation of Halloysite for Use as a Microtubular Drug Delivery System. Int. J. Pharm. 2002, 243, 125–134.

(16)

Krishnan, S. K.; Rangel, A. H.; Pal, U.; Sánchez, O. C.; Flores-Ruiz, F. J.; Prokhorov, Y.; Arias de Fuentes, O.; Esparza, R.; Meyyappan, M. Surface Functionalized Halloysite Nanotubes Decorated with Silver Nanoparticles for Enzyme Immobilization and Biosensing. J. Mater. Chem. B 2016, 4, 2553–2560.

(17)

Chao, C.; Zhang, B.; Zhai, R.; Xiang, X.; Liu, J.; Chen, R. Natural Nanotube-Based Biomimetic

Porous

Microspheres

for

Significantly

Enhanced

Biomolecule

Immobilization. ACS Sustainable Chem. Eng. 2014, 2, 396–403. (18)

Yang, T.; Du, M.; Zhang, M.; Zhu, H.; Wang, P.; Zou, M. Synthesis and Immobilization of Pt Nanoparticles on Amino-Functionalized Halloysite Nanotubes toward Highly Active Catalysts. Nanomater. Nanotechnol. 2015, 5, 1-9.

(19)

Yuan, P.; Southon, P. D.; Liu, Z.; Kepert, C. J. Organosilane Functionalization of Halloysite Nanotubes for Enhanced Loading and Controlled Release. Nanotechnology 29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 34

2012, 23, 375705. (20)

Zhang, Y.; He, X.; Ouyang, J.; Yang, H. Palladium Nanoparticles Deposited on Silanized Halloysite Nanotubes: Synthesis, Characterization and Enhanced Catalytic Property. Sci. Rep. 2013, 3, 2948.

(21)

Yuan, P.; Southon, P. D.; Liu, Z.; Malcolm, E. R.; Hook, J. M.; Antill, S. J.; Kepert, C. J.; Functionalization

of

Halloysite

Clay

Nanotubes

by

Grafting

with

γ-

Aminopropyltriethoxysilane. J. Phys. Chem. C 2008, 112, 15742–15751. (22)

Luo, P.; Zhang, J. S.; Zhang, B.; Wang, J. H.; Zhao, Y. F.; Liu, J. D. Preparation and Characterization of Silane Coupling Agent Modified Halloysite for Cr(VI) Removal. Ind. Eng. Chem. Res. 2011, 50, 10246–10252.

(23)

Zhang, Y.; Xie, Y.; Tang, A.; Zhou, Y.; Ouyang, J.; Yang, H. Precious-Metal Nanoparticles Anchored onto Functionalized Halloysite Nanotubes. Ind. Eng. Chem. Res. 2014, 53, 5507–5514.

(24)

An, N.; Zhou, C. H.; Zhuang, X. Y.; Tong, D. S.; Yu, W. H. Immobilization of Enzymes on Clay Minerals for Biocatalysts and Biosensors. Appl. Clay Sci. 2015, 114, 283–296.

(25)

Pan, J.; Yao, H.; Xu, L.; Ou, H.; Huo, P.; Li, X.; Yan, Y. Selective Recognition of 2,4,6Trichlorophenol by Molecularly Imprinted Polymers Based on Magnetic Halloysite Nanotubes Composites. J. Phys. Chem. C 2011, 115, 5440–5449.

(26)

Bradford, M. M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 1976, 72, 248–254.

(27)

Sahu, A.; Badhe, P. S.; Adivarekar, R.; Ladole, M. R.; Pandit, A. B. Synthesis of Glycinamides Using Protease Immobilized Magnetic Nanoparticles. Biotechnol. Rep. 30


Page 31 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2016, 12, 13–25. (28)

Dai, Y.; Niu, J.; Yin, L.; Xu, J.; Xu, J. Laccase-Carrying Electrospun Fibrous Membrane for the Removal of Polycyclic Aromatic Hydrocarbons from Contaminated Water. Sep. Purif. Technol. 2013, 104, 1–8.

(29)

Zhai, R.; Zhang, B.; Liu, L.; Xie, Y.; Zhang, H.; Liu, J. Immobilization of Enzyme Biocatalyst on Natural Halloysite Nanotubes. Catal. Commun. 2010, 12, 259–263.

(30)

Yao, J.; Wang, Q.; Wang, Y.; Zhang, Y.; Zhang, B.; Zhang, H. Immobilization of Laccase on Chitosan-Halloysite Hybrid Porous Microspheres for Phenols Removal. Desalin. Water Treat. 2015, 55, 1293–1301.

(31)

Xin, B.; Si, S.; Xing, G. Protease Immobilization on Gamma-Fe2O3/Fe3O4 Magnetic Nanoparticles for the Synthesis of Oligopeptides in Organic Solvents. Chem. Asian J. 2010, 5, 1389–1394.

(32)

Song, C.; Sheng, L.; Zhang, X. Immobilization and Characterization of a Thermostable Lipase. Mar. Biotechnol. 2013, 15, 659–667.

(33)

Hu, T. G.; Cheng, J. H.; Zhang, B. B.; Lou, W. Y.; Zong, M. H. Immobilization of Alkaline Protease on Amino-Functionalized Magnetic Nanoparticles and Its Efficient Use for Preparation of Oat Polypeptides. Ind. Eng. Chem. Res. 2015, 54, 4689–4698.

(34)

Homaei, A. A.; Sariri, R.; Vianello, F.; Stevanato, R. Enzyme Immobilization: An Update. J. Chem. Biol. 2013, 6, 185–205.

(35)

Betancor, L.; Lopez-Gallego, F.; Hidalgo, A.; Alonso-Morales, N.; Dellamora-Ortiz, G.; Mateo, C.; Fernandez-Lafuente, R.; Guisan, J. M. Different Mechanisms of Protein Immobilization on Glutaraldehyde Activated Supports: Effect of Support Activation and Immobilization Conditions. Enzyme Microb. Technol. 2006, 39, 877–882. 31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(36)

Page 32 of 34

Schoevaart, R.; Wolbers, M. W.; Golubovic, M.; Ottens, M.; Kieboom, A. P. G.; Van Rantwijk, F.; Van Der Wielen, L. A. M.; Sheldon, R. A. Preparation, Optimization, and Structures, of Cross-Linked Enzyme Aggregates (CLEAs). Biotechnol. Bioeng. 2004, 87, 754–762.

(37)

Erdogan, A. R.; Kaygusuz, I.; Kaynak, C. Influences of Aminosilanization of Halloysite Nanotubes on the Mechanical Properties of Polyamide-6 Nanocomposites. Polym. Polym. Compos. 2014, 35, 1350–1361.

(38)

Zhong, S.; Zhou, C.; Zhang, X.; Zhou, H.; Li, H.; Zhu, X.; Wang, Y. A Novel Molecularly Imprinted Material Based on Magnetic Halloysite Nanotubes for Rapid Enrichment of 2,4Dichlorophenoxyacetic Acid in Water. J. Hazard. Mater. 2014, 276, 58–65.

(39)

Mohiuddin, M.; Arbain, D.; Islam, A. K. M.; Rahman, M.; Ahmad, M. S.; Ahmad, M. N. Covalent Immobilization of α-Glucosidase Enzyme onto Amine Functionalized MultiWalled Carbon Nanotubes. Curr. Nanosci. 2014, 10, 730–735.

(40) Awad, G. E. A.; Abd El Aty, A. A.; Shehata, A. N.; Hassan, M. E.; Elnashar, M. M. Covalent Immobilization of Microbial Naringinase Using Novel Thermally Stable Biopolymer for Hydrolysis of Naringin. 3 Biotech. 2016, 6, 14. (41)

Deng, S.; Zhang, J.; Ye, L.; Wu, J. Toughening Epoxies with Halloysite Nanotubes. Polymer. 2008, 49, 5119–5127.

(42)

Du, M.; Guo, B.; Jia, D. Thermal Stability and Flame Retardant Effects of Halloysite Nanotubes on Poly(propylene). Eur. Polym. J. 2006, 42, 1362–1369.

(43)

Kadam, A. A.; Jang, J.; Lee, D. S. Facile Synthesis of Pectin-Stabilized Magnetic Graphene Oxide Prussian Blue Nanocomposites for Selective Cesium Removal from Aqueous Solution. Bioresour. Technol. 2016, 216, 391–398. 32


Page 33 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(44)

Xu, W.; Riikonen, J.; Nissinen, T.; Suvanto, M.; Rilla, K.; Li, B.; Wang, Q.; Deng, F.; Lehto, V. P. Amine Surface Modifications and Fluorescent Labeling of Thermally Stabilized Mesoporous Silicon Nanoparticles. J. Phys. Chem. C 2012, 116, 22307–22314.

(45)

Liu, Y.; Zeng, Z.; Zeng, G.; Tang, L.; Pang, Y.; Li, Z.; Liu, C.; Lei, X.; Wu, M.; Ren, P.; Liu, Z.; Chen, M.; Xie, G. Immobilization of Laccase on Magnetic Bimodal Mesoporous Carbon and the Application in the Removal of Phenolic Compounds. Bioresour. Technol. 2012, 115, 21–26.

(46)

Rahmani, K.; Faramarzi, M. L.; Mahvi, A. H.; Gholami, M.; Esrafili, A.; Forootanfar, H.; Farzadkia, M. Elimination and Detoxification of Sulfathiazole and Sulfamethoxazole Assisted by Laccase Immobilized on Porous Silica Beads. Int. Biodeterior. Biodegrad. 2015, 97, 107-114.

(47)

Zhai, R.; Zhang, B.; Wan, Y.; Li, C.; Wang, J.; Liu, J. Chitosan-Halloysite HybridNanotubes: Horseradish Peroxidase Immobilization and Applications in Phenol Removal. Chem. Eng. J. 2013, 214, 304–309.

(48)

Margot, J.; Maillard, J.; Rossi, L.; Barry, D. A.; Holliger, C. Influence of Treatment Conditions on the Oxidation of Micropollutants by Trametes versicolor Laccase. New Biotechnol. 2013, 30, 803-813.

(49) Wong, K. S.; Cheung, M. K.; Au, C. H.; Kwan, H. S. A Novel Lentinula Edodes Laccase and Its Comparative Enzymology Suggest Guaiacol-Based Laccase Engineering for Bioremediation. PLoS One 2013, 8, 1–12.

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Page 34 of 34

TABLE OF CONTENTS GRAPHIC

SMX Guaiacol, ABTS Syringaldehyde

Fe3O4 HNTs

APTES

M-HNTs

Laccase

GTA

A-M-HNTs

A-M-HNTs-GTA A-M-HNTs-GTA-Lac

34


Supermagnetically Tuned Halloysite Nanotubes ... - ACS Publications

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