Next Article in Journal
Development of Novel Polypropylene Syntactic Foams Containing Paraffin Microcapsules for Thermal Energy Storage Applications
Next Article in Special Issue
Preparation of Coupling Catalyst HamZIF-90@Pd@CALB with Tunable Hollow Structure for Efficient Dynamic Kinetic Resolution of 1-Phenylethylamine
Previous Article in Journal
[MII(H2dapsc)]-[Cr(CN)6] (M = Mn, Co) Chain and Trimer Complexes: Synthesis, Crystal Structure, Non-Covalent Interactions and Magnetic Properties
Previous Article in Special Issue
Lipase-Catalyzed Phospha-Michael Addition Reactions under Mild Conditions
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Molecules
Volume 27
Issue 23
10.3390/molecules27238522
molecules-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Magnetic Polyethyleneimine Nanoparticles Fabricated via Ionic Liquid as Bridging Agents for Laccase Immobilization and Its Application in Phenolic Pollutants Removal

by
Runtang Liu
,
Wei Zhang
,
Shushu Wang
,
Huajin Xu
and
Yi Hu
*
State Key Laboratory of Materials-Oriented Chemical Engineering, School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing 210009, China
*
Author to whom correspondence should be addressed.
Molecules 2022, 27(23), 8522; https://doi.org/10.3390/molecules27238522
Submission received: 17 November 2022 / Revised: 29 November 2022 / Accepted: 1 December 2022 / Published: 3 December 2022
(This article belongs to the Special Issue Modern Trends of Biocatalysis in Organic Chemistry and Enzyme Engineering)
Browse Figures
Versions Notes

Abstract

:
In this study, polyethyleneimine was combined with magnetic Fe3O4 nanoparticles through the bridging of carboxyl-functionalized ionic liquid, and laccase was loaded onto the carrier by Cu2+ chelation to achieve laccase immobilization (MCIL–PEI–Cu–lac). The carrier was characterized by Fourier transform infrared spectroscopy, scanning electron microscope, thermogravimetric analysis, X-ray diffraction analysis, magnetic hysteresis loop and so on. MCIL–PEI–Cu–lac has good immobilization ability; its loading and activity retention could reach 52.19 mg/g and 91.65%, respectively. Compared with free laccase, its thermal stability and storage stability have been significantly improved, as well. After 6 h of storage at 60 °C, 51.45% of the laccase activity could still be retained, and 81.13% of the laccase activity remained after 1 month of storage at 3 °C. In the pollutants removal test, the removal rate of 2,4-dichlorophenol (10 mg/L) by MCIL–PEI–Cu–lac could reach 100% within 10 h, and the removal efficiency could still be maintained 60.21% after repeated use for 8 times. In addition, MCIL–PEI–Cu–lac also has a good removal effect on other phenolic pollutants (such as bisphenol A, phenol, 4-chlorophenol, etc.). Research results indicated that an efficient strategy for laccase immobilization to biodegrade phenolic pollutants was developed.

1. Introduction

Phenolic compounds are widely used in the industrial fields of fine chemicals production such as medicines, pesticides, dyes and daily chemicals, while this also leads to a large amount of phenolic pollutants in the wastewater generated in these fields [1,2,3]. These phenolic pollutants cause serious harm to nature and human health [4,5]. How to effectively remove pollutants from wastewater has become one of the key concerns in the environmental field.
The traditional treatment methods for phenolic pollutants mainly include physical adsorption and chemical catalysis [6,7], but these methods usually have the disadvantages of low efficiency and easy to cause secondary pollution. The enzymatic method is considered to be an effective method for degrading phenolic pollutants due to its high specificity, environmental friendliness and high catalytic efficiency [8,9]. Laccase is a kind of oxidoreductase with copper as the catalytic center. It has strong redox ability and a wide range of substrates, and the only by-product is water, which is very friendly to the environment [10,11]. However, free laccase will face a very complex environment when treating actual industrial wastewater, which can easily reduce the activity and stability of laccase [12]. In order to solve these problems, the development of a suitable immobilized enzyme carriers is of great significance to improve the thermal stability, storage stability, catalytic efficiency and reusability of laccase.
Up to now, the materials used to immobilize laccase include nanomaterials, membrane materials, microspheres and so on [13,14,15]. Among them, magnetic nanomaterials have the advantages of easy separation, good mechanical stability, easy surface modification, low toxicity and so on, which have been widely used in laccase immobilization [16,17]. However, individual magnetic material often has the disadvantages of easy agglomeration, low loading and low activity retention, so its surface needs to be modified to improve this phenomenon [18]. In recent years, various surface modification methods of magnetic materials have been developed, which solve these problems to a certain extent. However, it is difficult to greatly improve the immobilized enzyme activity, stability, reusability and other properties at the same time. For example, Ran [19] et al. used polyethyleneimine to directly coat magnetic materials for the immobilization of laccase. Although the loading of laccase reached 70 mg/g, the activity retention was only 80%. Meanwhile, Xia [20] et al. combined amino-functionalized magnetic nanoparticles (Fe3O4-NH2) with polyethyleneimine using glutaraldehyde as a crossing-linker (Fe3O4-NH2-PEI) to immobilize laccase via Cu2+ chelation. Although the loading of laccase is only 26.13 mg/g, its activity retention rate reaches 107.41%. The reason for the increased activity may be that Cu2+ coordination can reduce the deformation and desorption of laccase, and Cu2+ can activate laccase to a certain extent and enhance the activity of laccase [21,22].
Generally, ionic liquids composed of organic cations and anions could be used as green solvents and additives. In our previous study, functionalized ionic liquids were used as novel surface modifiers or bridging agents of organic–inorganic nanomaterials for porcine pancreatic lipase and laccase immobilization. Various enzymatic properties such as the enzyme loading, activity, stability and repeatability of the immobilized enzyme were effectively improved [23,24,25].
In this study, carboxyl functionalized ionic liquid was used as a bridging agent to combine magnetic Fe3O4 nanoparticles with polyethyleneimine, and laccase was immobilized on magnetic polyethyleneimine nanoparticles by copper ion chelation (Scheme 1). Subsequently, the removal ability of immobilized laccase for phenolic pollutants was investigated. We wish to combine the advantages of magnetic nanoparticles, functional ionic liquids, polyethyleneimine and Cu2+ activation to prepare an efficient carrier for laccase immobilization. We hope to propose an effective solution to the current bottleneck problem of laccase immobilization.

2. Results and Discussions

2.1. Analysis of Characterizations

Figure 1a shows the XRD characterization of Fe3O4, MPEI, MCIL and MCIL–PEI nanoparticles. The peaks at 2θ = 30.1°, 35.5°, 43.2°, 53.5°, 57.2° and 62.6° are consistent with the characteristic peaks of magnetic nanoparticles Fe3O4 (JCPDS = 77–1545) reported in the literature [26]. After further modification by carboxyl-functionalized ionic liquid and polyethyleneimine, carries still maintained the same peaks, and the peaks’ intensities were basically unchanged, which indicated that the crystal structure of carries was well preserved.
Figure 1b shows the Fe3O4, MCIL and MCIL–PEI FT-IR spectrum. In the Fe3O4 spectrum, the peak at 582 cm−1 is the characteristic peak of Fe3O4, and the peak at 3438 cm−1 proves that Fe3O4 is rich in hydroxyl groups [27]. In the MCIL spectrum, the absorption peak at 1727 cm−1 is attributed to the C = O stretching vibration of the carboxyl group, while the new peak at 1416 cm−1 is due to the C = C stretching vibration of the imidazole ring in the ionic liquid [28]. These peaks proved that Fe3O4 was successfully modified by carboxylated ionic liquids. In the MPEI spectrum, the characteristic peaks of polyethyleneimine were successfully detected, which proved that polyethyleneimine was successfully grafted to the surface of Fe3O4. In the MCIL–PEI spectrum, the broad peaks at 3500–3300 cm−1 could be attributed to the simultaneous presence of -NH2 and -NH groups in the material [29]. Furthermore, 1630 cm−1and 1536 cm−1 are the N–H bending vibration peak of primary and secondary amine, and 1059 cm−1 is the stretching vibration peak of C–N [30]. At the same time, the peak at 1421 cm−1 could be attributed to the C = C stretching vibration of the ionic liquid’s imidazole ring, which does not appear in the MPEI curve. This proves that the MCIL–PEI carrier contains an ionic liquid structure. The absorption peak at 891 cm−1 indicates the existence of a tertiary amine structure in MCIL–PEI [19]. The above results indicated that carboxyl-functionalized ionic liquid and PEI were successfully modified onto the surface of MCIL nanomaterials.
Thermogravimetric analysis of Fe3O4, MCIL and MCIL–PEI was performed, and Figure 2a shows the TGA curves of the samples. From the TGA curve of Fe3O4, the weight dropped 7.91%, which may be due to the evaporation of water adsorbed on Fe3O4. The TGA curve of MCIL dropped significantly after 300 °C, indicating that the grafted carboxylated ionic liquid structure undergoes rapid thermal decomposition, accounting for 11.47% of the total weight. The weight loss of the MPEI TGA curve before 200 °C should be the loss of moisture in the material. After 300 °C, the weight loss speed was accelerated, and finally, 74.47% of the weight remained. It showed that PEI was successfully grafted to the surface of Fe3O4, and MPEI was successfully synthesized. From the TGA curve of MCIL–PEI, it can be seen that when the temperature reached 200 °C, the decreasing speed of the total weight begins to accelerate. At the same time, when the temperature reached at 610 °C, the structure of the carboxyl-functionalized ionic liquid was completely decomposed. However, the weight was still decreasing at this time, indicating that the polyethyleneimine was not completely decomposed. The final total weight loss indicated that MCIL–PEI contained 18.31% polyethyleneimine. TGA analysis showed that magnetic nanoparticles were successfully modified with carboxylated ionic liquids and polyethyleneimine.
The magnetic hysteresis loops of carriers are exhibited in Figure 2b. Fe3O4 and MCIL–PEI–Cu nanoparticles exhibit excellent superparamagnetism [27], which is beneficial to the separation and reuse of immobilized laccase. The saturation magnetization of Fe3O4 and MCIL–PEI–Cu are 64.1 and 43.8 emu/g. The grafting of ionic liquid and polyvinyl imine reduced the magnetization of the carriers.
It can be observed from Figure 3 that the prepared Fe3O4 (a, c) is a nanoscale material with a particle size of 21.85 nm, but the agglomeration phenomenon is very obvious [31]. The modified carrier MCIL–PEI–Cu (b, d) was still spherical or elliptical, but the particle size of MCIL–PEI–Cu is 24.33 nm, which is improved compared with Fe3O4. At the same time, PEI is a water-soluble cationic polymer with high density of various amino functional groups [22,32]. It can prevent particle agglomeration by using electrostatic repulsion and steric hindrance [33,34]. The dispersibility of MCIL–PEI–Cu nanoparticles is greatly improved compared with Fe3O4 nanoparticles, indicating that polyethyleneimine has been successfully grafted on the surface of Fe3O4. From EDS characterization (Figure 4), in the EDS spectrum of MCIL–PEI–Cu, the newly added elements C, N, F and B indicate that the ionic liquid and PEI were successfully modified to the surface of magnetic nanoparticles, and the presence of Cu elements meant the successful introduction of Cu ions.

2.2. Results of Laccase Immobilization

As shown in Table 1, compared with MPEI, the loading capacity and activity retention of laccase immobilized on MCIL–PEI were improved to a certain extent. This indicated that the introduction of carboxyl-functionalized ionic liquid groups has a positive effect on the retention of laccase activity while increasing the enzyme loading capacity. The possible reason is that the ionic liquid structure provides a good microenvironment for the immobilization of laccase. Additionally, the introduction of ionic liquid can further enhance the interaction between the enzyme and the carrier, which is conducive to maintaining the integrity and activity of the enzyme conformation. Meanwhile, it has a strong hydrogen bonding ability, which better protects the spatial conformation of laccase. Compared with the above two carriers, MCIL–PEI–Cu has an obvious improvement in enzyme loading and activity retention. Obviously, the introduction of Cu2+ increases the action sites of the carrier through coordination, so that the laccase has more binding sites and greatly increases the laccase loading. On the other hand, the introduction of Cu2+ can significantly enhance the activity of immobilized laccase, and its activity retention rate is as high as 91.65%, showing a higher level of activity retention than previously reported in literature. Seyed Mehdi [35] et al. used aminated magnetic ferric oxide to immobilize laccase through glutaraldehyde cross-linking, retaining only 27% of its activity. Chen [36] et al. modified the surface of magnetic nanoparticles with polydopamine and immobilized laccase with dialdehyde starch as a cross-linking agent, but the activity retention was only 69%. It might be that Cu2+ had a certain activation effect on the active center of laccase, which was consistent with previous literature reports [37].

2.3. Stability Test

A stability test is also necessary to study whether the immobilized laccase has industrial application value. As shown in Figure 5a, the activity of free laccase decreased significantly at a high temperature of 60 °C, and only 12.31% of the enzyme activity remained after 6 h. The thermal stability of three immobilized laccases was greatly improved compared with free laccases. The best immobilized laccase, MCIL–PEI–Cu–lac, still retained 51.45% of the laccase activity after 6 h. In the storage stability test (Figure 5b), the free laccase only retained 40.21% of its enzyme activity after 30 days of storage. While MCIL–PEI–Cu–lac had the highest remaining activity after 30 days of storage, with a remaining activity of 81.13%; it also had a certain degree of improvement compared with other literature reports. Wu [38] et al. immobilized laccase with biomass-derived nanocellulose aerogels. After the immobilized laccase was stored at 4 °C for 20 days, the laccase activity remained at 66.4%. Taghizadeh [39] et al. immobilized laccase on sodium zeolite Y (NaY) and its modified desilicated (DSY) and dealuminated (DAY) forms, and the immobilized laccase activity remained at 83.7% after storage at 3 °C for 20 days. The reason for the improved stability may be the combined effect of various forces such as hydrogen bonding, electrostatic interaction and Cu2+ coordination [40]. They enable laccase to maintain the secondary structure of laccase in a high temperature environment, making laccase less prone to denaturation and inactivation.

2.4. DCP Removal Test

Figure 6a compares the removal efficiency of free laccase and MCIL–PEI–Cu–lac for 2,4-DCP (10 mg/L) in water. The maximum degradation efficiency (85.31%) of free laccase was reached at 10 h, and there was no significant improvement after a prolonged time, while MCIL–PEI–Cu–lac completely removed 2,4-DCP after 10 h. This confirms that the modification of PEI and the coordination of Cu2+ can not only improve the stability of the immobilized laccase MCIL–PEI–Cu–lac, but can also have excellent activity on the degradation of 2,4-DCP. The possible reason is that the abundant amino groups of PEI can form coordination bonds with laccase through the metal ion Cu2+. The coordination of Cu2+ method can reduce the deformation and desorption of laccase, and Cu2+ can activate laccase to a certain extent and enhance the activity of laccase [40], so that the final removal efficiency of MCIL–PEI–Cu–lac is higher than that of free laccase.

2.5. Reusability Test

Reusability has important value in practical applications. Compared with other materials, the magnetic material can be easily separated from the solution with a magnet. As shown in Figure 6b, MCIL–PEI–Cu–lac completely degraded 2,4-DCP (10 mg/L) in the first three cycles. After 8 cycles, the removal rate of 2,4-DCP can still reach 60.21%. On the one hand, the reason may be that the polymers generated by the degradation of phenolic pollutants will adhere to the outer surface of the carrier, occupying the active sites of laccase and reducing the oxidative degradation ability of the immobilized laccase [6]. On the other hand, the reason may be that the laccase is exposed to room temperature for too long during use, resulting in a certain decrease in the activity of the laccase [41]. At the same time, MCIL–PEI-Cu has better repeatability compared with other literature. Yang [42] et al. immobilized laccase on caged mesoporous SiO2 wrapped with chitosan/alginate microcapsule membrane. After 6 times of use, immobilized laccase could only remove about 50% of 2,4-DCP pollutant. Erol Alver [43] et al. immobilized laccase onto metal-chelated copolymer NPs retained about 60.0% of initial activity after 6 reaction cycles.

2.6. Expanding Applications Test

In order to further study the removal ability of immobilized laccase for other phenolic pollutants, the removal effects of immobilized laccase on 2,4-dichlorophenol (50 mg/L), bisphenol A, 4-chlorophenol and phenol (all 10 mg/L) were investigated (Figure 7). The results showed that when the removal concentration reached the 2,4-dcp concentration of 50 mg/L, the immobilized laccase still had a removal efficiency of 78.39% within 24 h. At the same time, when dealing with other phenolic pollutants, the removal effect of bisphenol A is the best, which can reach 88.51%. The treatment effect of 4-CP and phenol can also reach about 80%, which is also greatly improved compared with free laccase. Lin [44] et al. immobilized laccase onto Cu(II)- and Mn(II)-chelated magnetic microspheres and successfully applied to remove bisphenol A from water, and the maximum removal efficiency was 85.0%. Mohammadi [45] et al. immobilized laccase onto epoxy-functionalized silica particles and eventually removed 60% of 4-CP.

3. Materials and Methods

3.1. Materials

Laccase from Aspergillus oryzae was purchased by SUNSON (Ningxia, China). FeCl3·6H2O, FeCl2·4H2O, NH3·H2O (28%), 4-chlorophenol (4-CP), bisphenol A, 2,4-dichlorophenol (2,4-DCP) and phenol were provided by Aladdin (Shanghai, China). In addition, 3-chloropropyltrimethylsilane (CPTMO), 2,2-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and NaBF4 were supported by China National Pharmaceutical Group Corporation (Beijing, China). Chloroacetic acid and polyethylene imine (PEI) were purchased by Energy Chemical (Shanghai, China).

3.2. Characterizations

XRD analysis was performed on a Bruker D8 Advance instrument (Germany). FT-IR spectroscopy was obtained by a Bruker Vertex 70 FT-IR spectrometer in the range from 400 to 4000 cm−1 (Germany). VSM magnetic hysteresis loops were obtained on an MPMS XL-7 vibrating sample magnetometer. TGA was obtained on a Netzsch TG-209-F3 Nevio (Germany). SEM was conducted on a ZEISS Genimi500 instrument (Germany).

3.3. Preparation of Fe3O4 Magnetic Nanoparticles

The preparation of magnetic nano Fe3O4 refers to the previous literature [46] and is slightly modified. Firstly, 5.11 g FeCl3·6H2O and 1.99 g FeCl2·4H2O were dissolved into 100 mL deionized water and reacted at 70 °C under N2 protection. Then, the pH of the system was adjusted with 28% ammonia until the solution turned black completely, and reacted at 80 °C for 1.5 h. Finally, Fe3O4 particles generated by the reaction were collected and washed with deionized water until neutral. The final product was dried in a vacuum drying oven for 12 h.

3.4. Modification of Magnetic Fe3O4 Nanoparticles by Carboxylated Ionic Liquid

The magnetic Fe3O4 nanoparticles modified by carboxylated ionic liquids were prepared with reference to previous literature reports, and some modifications were made [24]. First, 3.0 g Fe3O4 nanoparticles were dispersed in toluene, 15 mmol 3-chloropropyltrimethylsilane (CPTMO) was added dropwise, reacted at 95 °C for 7 h under nitrogen protection, washed with ethanol and dried. After that, the obtained solid was dispersed with imidazole (15 mmol) in chloroform. After reacting at 35 °C under the protection of nitrogen for 24 h, the solid was separated by a magnet, washed with ethanol and dried. The intermediate product was then alkylated with 15 mmol of chloroacetic acid in acetonitrile at 50 °C for 12 h, separated by a magnet, washed with ethanol and dried. Finally, the intermediate product was anion-exchanged with NaBF4 in acetone for 48 h, separated by a magnet and washed with ethanol. The final product was dried in a vacuum oven for 24 h and designated as MCIL.

3.5. Preparation of Magnetic Polyethyleneimine Nanoparticles

First, 0.1 mol EDC, 0.1 mol NHS and 1.0 g MCIL were dispersed in 50 mL of citrate buffer solution (pH 7.0) and stirred at room temperature for 2 h. Then, 30 mL of PEI solution (2.5% w/v) was added, reacted at room temperature for 12 h, separated by magnet and washed with water and ethanol several times. The final product was dried in a vacuum oven for 12 h and designated as MCIL–PEI.
At the same time, in order to verify the effect of the incorporation of ionic liquid on the immobilization ability of the carrier, a comparative carrier was designed. The steps are as follows: 3.0 g Fe3O4 nanoparticles were dispersed in toluene, 15 mmol 3-chloropropyltrimethylsilane (CPTMO) was added dropwise, reacted at 95 °C for 7 h under nitrogen protection, separated by a magnet, washed several times with ethanol and dried. Afterwards, the intermediate product and 30 mL of PEI solution (2.5% w/v) were reacted at room temperature for 12 h, separated by a magnet and washed with ethanol several times. The final product was dried in a vacuum oven for 24 h and designated as MPEI.
Referring to the method reported in the previous literature [37], we introduced Cu2+ on MCIL–PEI. Then, 1.0 g of MCIL–PEI was dispersed in 25 mL of acetonitrile solution and 15 mmol of CuCl2 were added. The reaction was carried out under N2 protection for 48 h, and the solid was separated with a magnet and washed with deionized water. The product was designated as MCIL–PEI–Cu.

3.6. Laccase Activity Assay

Laccase activity was determined by measuring the rate of oxidation of ABTS by free and immobilized laccase [37,47]. An appropriate amount of laccase was added to 5 mL (1 mM) of citrate buffer at different pH, and the reaction was carried out at different temperatures for 5 min. The absorbance of the supernatant was measured by UV–vis spectrophotometer at 420 nm. One unit of laccase activity (1 U) was defined as the amount of laccase required to catalyze the oxidation of 1 μmol of ABTS in one minute under certain conditions. Each test was performed three times, and the average taken. The laccase activity was calculated according to the following formula:
Expressed activity (U/g biocatalyst) = A × 106 × Vt/(36000 × t × m1)
Specific activity (U/g protein) = A × 106 × Vt/(36000 × t × m2)
A: absorbance at 420 nm; Vt: total volume (L); 36,000: molar extinction coefficient of ABTS·+ (M−1 cm−1); t: reaction time (min); m1: quality of immobilized laccase (g); m2: protein quality of immobilized laccase (g).

3.7. Laccase Immobilization

The optimization of enzyme immobilization process conditions is an important factor in the preparation of enzyme immobilization [48]. Enzyme loading and activity retention are important parameters for immobilized enzymes. We investigated the immobilization effect of MPEI, MCIL–PEI and MCIL–PEI–Cu under different conditions of enzyme concentration, immobilization time, pH and temperature (Figures S1–S4). Furthermore, the laccase immobilization process was optimized using protein loading and activity retention as indicators.
Laccase was immobilized on the three carriers MPEI, MCIL–PEI and MCIL–PEI–Cu under their respective optimum conditions, and the immobilized laccases were named MPEI–lac, MCIL–PEI–lac and MCIL–PEI–Cu–lac. Immobilized laccases were collected with a magnet and stored lyophilized. The determination of protein content in the supernatant was performed by the Bradford [49] method, each result performed three times, and the average taken.

3.8. Stability Test

Stability is an important indicator to evaluate the industrial application of laccase [50]. During the determination of thermal stability, the free and immobilized laccases were placed at 60 °C for some time, and the laccase activities were measured every hour, defining the initial laccase activity to 100%.
At the same time, in order to determine the storage stability, various laccases were stored at 3 °C for a month. Laccase activity was measured every 5 days, defining the initial laccase activity to 100%.

3.9. Phenolics Removal Test

In order to study the removal effect of free laccase and immobilized laccase on 2,4-DCP (10 mg/L), an appropriate amount (1.5 U) of free laccase and immobilized laccase was added to 10 mL 2,4-DCP aqueous solution, respectively, and reacted at room temperature at 12 h. The liquid chromatography measurement conditions are as follows: the mobile phase, wavelength and flow rate were 70% methanol in water, 220 nm and 1 mL/min for 10 min, respectively. Each result was measured in triplicate and averaged. The 2,4-DCP removal efficiency formula is as follows:
RE (%) = (A0 − At)/A0 × 100
A0: corresponding peak area before removal; At: corresponding peak area after removal.
In addition, the removal efficiency of MCIL–PEI–Cu–lac for other phenolic pollutants 2,4-DCP (50 mg/L), bisphenol A (10 mg/L), 4-chlorophenol (10 mg/L) and phenol (10 mg/L) was also determined, and the reaction was carried out at room temperature for 24 h. Bisphenol A measurement conditions: the mobile phase, wavelength and flow rate were 70% methanol in water, 290 nm and 1 mL/min for 10 min, respectively. The 4-chlorophenol and phenol measurement conditions were: the mobile phase, wavelength and flow rate were 70% methanol in water, 220 nm and 1 mL/min for 10 min, respectively.

3.10. Reusability Test

To determine the reusability of immobilized laccase, 1.5 U of immobilized laccase was treated with 10 mL 2,4-DCP (10 mg/L) in water at room temperature for 10 h. Afterwards, the immobilized laccase was collected with a magnet and washed three times with deionized water. The above experimental procedure was repeated after lyophilization to test the reusability.

4. Conclusions

In this study, polyethyleneimine PEI was combined with magnetic nanoparticles through carboxyl-functionalized ionic liquid as a bridge agent. Then laccase was immobilized on the carrier through the coordination with Cu2+, which has an activating effect on laccase. The results have shown that the enzyme loading and activity retention rate of MCIL–PEI–Cu–lac could reach 52.19 mg/g and 91.65%, respectively. At the same time, it has excellent thermal stability and storage stability. The laccase activity remained at 51.45% after storage at 60 °C for 6 h, and 81.13% after storage at 3 °C for 1 month. The removal rate of 2,4-DCP (10 mg/L) by MCIL–PEI–Cu–lac can reach 100%, and the removal rate remains at 60.21% after repeated use for 8 times. In addition, MCIL–PEI–Cu–lac also had a good removal effect on bisphenol A, phenol, and 4-chlorophenol. This biocatalyst has good application value in removing phenolic pollutants, and it is beneficial to the further application of the immobilized laccase in the treatment of phenol-containing wastewater.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules27238522/s1, Figure S1: Effect of time on laccase immobilization; Figure S2: Effect of enzyme concentration on laccase immobilization; Figure S3: Effect of pH on laccase immobilization; Figure S4: Effect of temperature on laccase immobilization.

Author Contributions

Conceptualization, R.L.; methodology, R.L., W.Z., S.W. and H.X.; formal analysis, R.L.; investigation, R.L., W.Z., S.W. and H.X.; data curation, R.L.; writing—original draft, R.L.; writing—review and editing, R.L. and Y.H.; funding acquisition, Y.H.; supervision, Y.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financially supported by the National Natural Science Foundation of China (No. 22178174) and the National Key R&D program of China (No. 2021YFC2103800), as well as The Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture (No. XTC2206).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

This research was financially supported by the National Natural Science Foundation of China (No. 22178174) and the National Key R&D program of China (No. 2021YFC2103800), as well as The Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture (No. XTC2206).

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Olaniran, A.O.; Igbinosa, E.O. Chlorophenols and other related derivatives of environmental concern: Properties, distribution and microbial degradation processes. Chemosphere 2011, 83, 1297–1306. [Google Scholar] [CrossRef] [PubMed]
  2. Torres, E.; Bustos-Jaimes, I.; Le Borgne, S. Potential use of oxidative enzymes for the detoxification of organic pollutants. Appl. Catal. B-Environ. 2003, 46, 1–15. [Google Scholar] [CrossRef]
  3. Das, T.K.; Das, N.C. Advances on catalytic reduction of 4-nitrophenol by nanostructured materials as benchmark reaction. Int. Nano Lett. 2022, 12, 223–242. [Google Scholar] [CrossRef]
  4. Furukawa, K. Oxygenases and dehalogenases: Molecular approaches to efficient degradation of chlorinated environmental pollutants. Biosci. Biotechnol. Biochem. 2006, 70, 2335–2348. [Google Scholar] [CrossRef] [PubMed]
  5. Das, T.K.; Remanan, S.; Ghosh, S.; Das, N.C. An environment friendly free-standing cellulose membrane derived for catalytic reduction of 4-nitrophenol: A sustainable approach. J. Environ. Chem. Eng. 2021, 9, 104596. [Google Scholar] [CrossRef]
  6. Couto, S.R.; Herrera, J.L.T. Industrial and biotechnological applications of laccases: A review. Biotechnol. Adv. 2006, 24, 500–513. [Google Scholar] [CrossRef]
  7. Kim, K.H.; Ihm, S.K. Heterogeneous catalytic wet air oxidation of refractory organic pollutants in industrial wastewaters: A review. J. Hazard. Mater. 2011, 186, 16–34. [Google Scholar] [CrossRef]
  8. Alcalde, M.; Ferrer, M.; Plou, F.J. Environmental biocatalysis: From remediation with enzymes to novel green processes. Biocatal. Biotransformation 2007, 25, 113. [Google Scholar] [CrossRef]
  9. Khalid, N.; Kalsoom, U.; Ahsan, Z.; Bilal, M. Non-magnetic and magnetically responsive support materials immobilized peroxidases for biocatalytic degradation of emerging dye pollutants-A review. Int. J. Biol. Macromol. 2022, 207, 387–401. [Google Scholar] [CrossRef]
  10. Riva, S. Laccases: Blue enzymes for green chemistry. FEBS J. 2013, 280, 590. [Google Scholar] [CrossRef]
  11. Duran, N.; Rosa, M.A.; D’Annibale, A.; Gianfreda, L. Applications of laccases and tyrosinases (phenoloxidases) immobilized on different supports: A review. Enzym. Microb. Technol. 2002, 31, 907–931. [Google Scholar] [CrossRef]
  12. Lu, J.W.; Nie, M.F.; Li, Y.R.; Zhu, H.L.; Shi, G.Y. Design of composite nanosupports and applications thereof in enzyme immobilization: A review. Colloids Surf. B-Biointerfaces 2022, 217, 112602. [Google Scholar] [CrossRef]
  13. Hou, J.W.; Dong, G.X.; Ye, Y.; Chen, V. Enzymatic degradation of bisphenol-A with immobilized laccase on TiO2 sol-gel coated PVDF membrane. J. Membr. Sci. 2014, 469, 19–30. [Google Scholar] [CrossRef]
  14. Xu, R.; Si, Y.F.; Wu, X.T.; Li, F.T.; Zhang, B.R. Triclosan removal by laccase immobilized on mesoporous nanofibers: Strong adsorption and efficient degradation. Chem. Eng. J. 2014, 255, 63–70. [Google Scholar] [CrossRef]
  15. Sheldon, R.A.; van Pelt, S. Enzyme immobilisation in biocatalysis: Why, what and how. Chem. Soc. Rev. 2013, 42, 6223–6235. [Google Scholar] [CrossRef] [Green Version]
  16. Rossi, L.M.; Costa, N.J.S.; Silva, F.P.; Wojcieszak, R. Magnetic nanomaterials in catalysis: Advanced catalysts for magnetic separation and beyond. Green Chem. 2014, 16, 2906–2933. [Google Scholar] [CrossRef]
  17. Zhang, K.; Yang, W.Z.; Liu, Y.; Zhang, K.G.; Chen, Y.; Yin, X.S. Laccase immobilized on chitosan-coated Fe3O4 nanoparticles as reusable biocatalyst for degradation of chlorophenol. J. Mol. Struct. 2020, 1220, 128769. [Google Scholar] [CrossRef]
  18. Wang, X.Y.; Jiang, X.P.; Li, Y.; Zeng, S.; Zhang, Y.W. Preparation Fe3O4@chitosan magnetic particles for covalent immobilization of lipase from Thermomyces lanuginosus. Int. J. Biol. Macromol. 2015, 75, 44–50. [Google Scholar] [CrossRef]
  19. Ran, F.P.; Zou, Y.L.; Xu, Y.X.; Liu, X.Y.; Zhang, H.X. Fe3O4@MoS2@PEI-facilitated enzyme tethering for efficient removal of persistent organic pollutants in water. Chem. Eng. J. 2019, 375, 121947. [Google Scholar] [CrossRef]
  20. Xia, T.T.; Liu, C.Z.; Hu, J.H.; Guo, C. Improved performance of immobilized laccase on amine-functioned magnetic Fe3O4 nanoparticles modified with polyethylenimine. Chem. Eng. J. 2016, 295, 201–206. [Google Scholar] [CrossRef]
  21. Su, J.; Fu, J.J.; Wang, Q.; Silva, C.; Cavaco-Paulo, A. Laccase: A green catalyst for the biosynthesis of poly-phenols. Crit. Rev. Biotechnol. 2018, 38, 294–307. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Chen, B.; Zhao, X.S.; Liu, Y.; Xu, B.G.; Pan, X.J. Highly stable and covalently functionalized magnetic nanoparticles by polyethyleneimine for Cr(VI) adsorption in aqueous solution. RSC Adv. 2015, 5, 1398–1405. [Google Scholar] [CrossRef]
  23. Xiang, X.R.; Ding, S.; Suo, H.B.; Xu, C.; Gao, Z.; Hu, Y. Fabrication of chitosan-mesoporous silica SBA-15 nanocomposites via functional ionic liquid as the bridging agent for PPL immobilization. Carbohydr. Polym. 2018, 182, 245–253. [Google Scholar] [CrossRef] [PubMed]
  24. Qiu, X.; Qin, J.; Xu, M.; Kang, L.F.; Hu, Y. Organic-inorganic nanocomposites fabricated via functional ionic liquid as the bridging agent for Laccase immobilization and its application in 2,4-dichlorophenol removal. Colloids Surf. B-Biointerfaces 2019, 179, 260–269. [Google Scholar] [CrossRef] [PubMed]
  25. Qiu, X.; Wang, Y.; Xue, Y.; Li, W.X.; Hu, Y. Laccase immobilized on magnetic nanoparticles modified by amino-functionalized ionic liquid via dialdehyde starch for phenolic compounds biodegradation. Chem. Eng. J. 2020, 391, 123564. [Google Scholar] [CrossRef]
  26. Nguyen, X.S.; Zhang, G.K.; Yang, X.F. Mesocrystalline Zn-Doped Fe3O4 Hollow Submicrospheres: Formation Mechanism and Enhanced Photo-Fenton Catalytic Performance. ACS Appl. Mater. Interfaces 2017, 9, 8900–8909. [Google Scholar] [CrossRef]
  27. Suo, H.B.; Xu, L.L.; Xu, C.; Qu, X.; Chen, H.Y.; Huang, H.; Hu, Y. Graphene Oxide Nanosheets Shielding of Lipase Immobilized on Magnetic Composites for the Improvement of Enzyme Stability. ACS Sustain. Chem. Eng. 2019, 7, 4486–4494. [Google Scholar] [CrossRef]
  28. Zou, B.; Hu, Y.; Cui, F.J.; Jiang, L.; Yu, D.H.; Huang, H. Effect of surface modification of low cost mesoporous SiO2 carriers on the properties of immobilized lipase. J. Colloid Interface Sci. 2014, 417, 210–216. [Google Scholar] [CrossRef]
  29. Esmaeilnejad-Ahranjani, P.; Kazemeini, M.; Singh, G.; Arpanaei, A. Amine-functionalized magnetic nanocomposite particles for efficient immobilization of lipase: Effects of functional molecule size on properties of the immobilized lipase. RSC Adv. 2015, 5, 33313–33327. [Google Scholar] [CrossRef] [Green Version]
  30. Jiang, H.L.; Kwon, J.T.; Kim, E.M.; Kim, Y.K.; Arote, R.; Jere, D.; Jeong, H.J.; Jang, M.K.; Nah, J.W.; Xu, C.X.; et al. Galactosylated poly(ethylene glycol)-chitosan-graft-polyethylenimine as a gene carrier for hepatocyte-targeting. J. Control. Release 2008, 131, 150–157. [Google Scholar] [CrossRef]
  31. Chen, M.; Xu, P.; Zeng, G.M.; Yang, C.P.; Huang, D.L.; Zhang, J.C. Bioremediation of soils contaminated with polycyclic aromatic hydrocarbons, petroleum, pesticides, chlorophenols and heavy metals by composting: Applications, microbes and future research needs. Biotechnol. Adv. 2015, 33, 745–755. [Google Scholar] [CrossRef]
  32. Motevalizadeh, S.F.; Khoobi, M.; Sadighi, A.; Khalilvand-Sedagheh, M.; Pazhouhandeh, M.; Ramazani, A.; Faramarzi, M.A.; Shafiee, A. Lipase immobilization onto polyethylenimine coated magnetic nanoparticles assisted by divalent metal chelated ions. J. Mol. Catal. B-Enzym. 2015, 120, 75–83. [Google Scholar] [CrossRef]
  33. Chen, C.T.; Wang, L.Y.; Ho, Y.P. Use of polyethylenimine-modified magnetic nanoparticles for highly specific enrichment of phosphopeptides for mass spectrometric analysis. Anal. Bioanal. Chem. 2011, 399, 2795–2806. [Google Scholar] [CrossRef]
  34. Chen, Y.L.; Pan, B.C.; Zhang, S.J.; Li, H.Y.; Lv, L.; Zhang, W.M. Immobilization of polyethylenimine nanoclusters onto a cation exchange resin through self-crosslinking for selective Cu(II) removal. J. Hazard. Mater. 2011, 190, 1037–1044. [Google Scholar] [CrossRef]
  35. Sadeghzadeh, S.; Nejad, Z.G.; Ghasemi, S.; Khafaji, M.; Borghei, S.M. Removal of bisphenol A in aqueous solution using magnetic cross -linked laccase aggregates from Trametes hirsuta. Bioresour. Technol. 2020, 306, 123169. [Google Scholar] [CrossRef]
  36. Chen, C.; Sun, W.; Lv, H.Y.; Li, H.; Wang, Y.B.; Wang, P. Spacer arm-facilitated tethering of laccase on magnetic polydopamine nanoparticles for efficient biocatalytic water treatment. Chem. Eng. J. 2018, 350, 949–959. [Google Scholar] [CrossRef]
  37. Qiu, X.; Wang, S.S.; Miao, S.S.; Suo, H.B.; Xu, H.J.; Hu, Y. Co-immobilization of laccase and ABTS onto amino-functionalized ionic liquid-modified magnetic chitosan nanoparticles for pollutants removal. J. Hazard. Mater. 2021, 401, 123564. [Google Scholar] [CrossRef]
  38. Wu, D.S.; Lv, P.F.; Feng, Q.; Jiang, Y.; Yang, H.R.; Alfred, M.; Wei, Q.F. Biomass-derived nanocellulose aerogel enable highly efficient immobilization of laccase for the degradation of organic pollutants. Bioresour. Technol. 2022, 356, 127311. [Google Scholar] [CrossRef]
  39. Taghizadeh, T.; Talebian-Kiakalaieh, A.; Jahandar, H.; Amin, M.; Tarighi, S.; Faramarzi, M.A. Biodegradation of bisphenol A by the immobilized laccase on some synthesized and modified forms of zeolite Y. J. Hazard. Mater. 2020, 386, 121950. [Google Scholar] [CrossRef]
  40. Chen, T.T.; Yang, W.J.; Guo, Y.L.; Yuan, R.J.; Xu, L.; Yan, Y.J. Enhancing catalytic performance of beta-glucosidase via immobilization on metal ions chelated magnetic nanoparticles. Enzym. Microb. Technol. 2014, 63, 50–57. [Google Scholar] [CrossRef]
  41. Gaitan, I.J.; Medina, S.C.; Gonzalez, J.C.; Rodriguez, A.; Espejo, A.J.; Osma, J.F.; Sarria, V.; Almeciga-Diaz, C.J.; Sanchez, O.F. Evaluation of toxicity and degradation of a chlorophenol mixture by the laccase produced by Trametes pubescens. Bioresour. Technol. 2011, 102, 3632–3635. [Google Scholar] [CrossRef] [PubMed]
  42. Yang, J.Y.; Huang, Y.; Yang, Y.X.; Yuan, H.M.; Liu, X.N. Cagelike mesoporous silica encapsulated with microcapsules for immobilized laccase and 2, 4-DCP degradation. J. Environ. Sci. 2015, 38, 52–62. [Google Scholar] [CrossRef] [PubMed]
  43. Alver, E.; Metin, A.U. Chitosan based metal-chelated copolymer nanoparticles: Laccase immobilization and phenol degradation studies. Int. Biodeterior. Biodegrad. 2017, 125, 235–242. [Google Scholar] [CrossRef]
  44. Lin, J.; Liu, Y.J.; Chen, S.; Le, X.Y.; Zhou, X.H.; Zhao, Z.Y.; Ou, Y.Y.; Yang, J.H. Reversible immobilization of laccase onto metal-ion-chelated magnetic microspheres for bisphenol A removal. Int. J. Biol. Macromol. 2016, 84, 189–199. [Google Scholar] [CrossRef] [PubMed]
  45. Mohammadi, M.; As’habi, M.A.; Salehi, P.; Yousefi, M.; Nazari, M.; Brask, J. Immobilization of laccase on epoxy-functionalized silica and its application in biodegradation of phenolic compounds. Int. J. Biol. Macromol. 2018, 109, 443–447. [Google Scholar] [CrossRef]
  46. Suo, H.B.; Xu, L.L.; Xu, C.; Chen, H.Y.; Yu, D.H.; Gao, Z.; Huang, H.; Hu, Y. Enhancement of catalytic performance of porcine pancreatic lipase immobilized on functional ionic liquid modified Fe3O(4)-Chitosan nanocomposites. Int. J. Biol. Macromol. 2018, 119, 624–632. [Google Scholar] [CrossRef]
  47. Chao, C.; Liu, J.D.; Wang, J.T.; Zhang, Y.W.; Zhang, B.; Zhang, Y.T.; Xiang, X.; Chen, R.F. Surface Modification of Halloysite Nanotubes with Dopamine for Enzyme Immobilization. ACS Appl. Mater. Interfaces 2013, 5, 10559–10564. [Google Scholar] [CrossRef]
  48. Suo, H.B.; Gao, Z.; Xu, L.L.; Xu, C.; Yu, D.H.; Xiang, X.R.; Huang, H.; Hu, Y. Synthesis of functional ionic liquid modified magnetic chitosan nanoparticles for porcine pancreatic lipase immobilization. Mater. Sci. Eng. C-Mater. Biol. Appl. 2019, 96, 356–364. [Google Scholar] [CrossRef]
  49. 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. [Google Scholar] [CrossRef]
  50. Chen, Z.H.; Yao, J.; Ma, B.; Liu, B.; Kim, J.; Li, H.; Zhu, X.Z.; Zhao, C.C.; Amde, M. A robust biocatalyst based on laccase immobilized superparamagnetic Fe3O4@SiO2-NH2 nanoparticles and its application for degradation of chlorophenols. Chemosphere 2022, 291, 132727. [Google Scholar] [CrossRef]
Molecules 27 08522 sch001 550
Scheme 1. The synthetic route map of magnetic polyethyleneimine nanoparticles.
Scheme 1. The synthetic route map of magnetic polyethyleneimine nanoparticles.
Molecules 27 08522 sch001
Molecules 27 08522 g001 550
Figure 1. XRD pattern (a) and FT-IR spectra (b) of carriers.
Figure 1. XRD pattern (a) and FT-IR spectra (b) of carriers.
Molecules 27 08522 g001
Molecules 27 08522 g002 550
Figure 2. TGA curve (a) and magnetic hysteresis loops (b) of carriers.
Figure 2. TGA curve (a) and magnetic hysteresis loops (b) of carriers.
Molecules 27 08522 g002
Molecules 27 08522 g003 550
Figure 3. SEM images of Fe3O4 (a,c) and MCIL–PEI–Cu (b,d).
Figure 3. SEM images of Fe3O4 (a,c) and MCIL–PEI–Cu (b,d).
Molecules 27 08522 g003
Molecules 27 08522 g004 550
Figure 4. EDS characterization of Fe3O4 (a) and MCIL–PEI–Cu (b).
Figure 4. EDS characterization of Fe3O4 (a) and MCIL–PEI–Cu (b).
Molecules 27 08522 g004
Molecules 27 08522 g005 550
Figure 5. Studies on thermal (a) and storage stability (b).
Figure 5. Studies on thermal (a) and storage stability (b).
Molecules 27 08522 g005
Molecules 27 08522 g006 550
Figure 6. 2,4-DCP removal test of various laccases (a). Reusability of MCIL–PEI–Cu–lac (b).
Figure 6. 2,4-DCP removal test of various laccases (a). Reusability of MCIL–PEI–Cu–lac (b).
Molecules 27 08522 g006
Molecules 27 08522 g007 550
Figure 7. Removal efficiency of other phenolic pollutants by free laccase and MCIL–PEI–Cu–lac.
Figure 7. Removal efficiency of other phenolic pollutants by free laccase and MCIL–PEI–Cu–lac.
Molecules 27 08522 g007
Table
Table 1. Results of laccase immobilization a.
Table 1. Results of laccase immobilization a.
CarriersLaccase ImmobilizationLaccase Activity Assay
Immobilization Efficiency (%)Enzyme Loading (mg g−1)Expressed Activity (U g−1 Biocatalyst)Specific Activity (U g−1 Protein)Activity Retention (%)
MPEI32.04 ± 0.0730.21 ± 0.0842.76 ± 0.151415.36 ± 3.4847.69 ± 0.14
MCIL–PEI36.26 ± 0.1134.17 ± 0.0958.67 ± 0.131717.01 ± 3.1457.85 ± 0.12
MCIL–PEI–Cu55.34 ± 0.1352.19 ± 0.12141.97 ± 0.162720.03 ± 3.6591.65 ± 0.16
a The activity of free laccase is 2967.84 U g−1 protein.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Liu, R.; Zhang, W.; Wang, S.; Xu, H.; Hu, Y. Magnetic Polyethyleneimine Nanoparticles Fabricated via Ionic Liquid as Bridging Agents for Laccase Immobilization and Its Application in Phenolic Pollutants Removal. Molecules 2022, 27, 8522. https://doi.org/10.3390/molecules27238522

AMA Style

Liu R, Zhang W, Wang S, Xu H, Hu Y. Magnetic Polyethyleneimine Nanoparticles Fabricated via Ionic Liquid as Bridging Agents for Laccase Immobilization and Its Application in Phenolic Pollutants Removal. Molecules. 2022; 27(23):8522. https://doi.org/10.3390/molecules27238522

Chicago/Turabian Style

Liu, Runtang, Wei Zhang, Shushu Wang, Huajin Xu, and Yi Hu. 2022. "Magnetic Polyethyleneimine Nanoparticles Fabricated via Ionic Liquid as Bridging Agents for Laccase Immobilization and Its Application in Phenolic Pollutants Removal" Molecules 27, no. 23: 8522. https://doi.org/10.3390/molecules27238522

Article Metrics

Back to TopTop