Skip to main content
Collagen and Leather
Download PDF

Laccase-functionalized magnetic framework composite enabled chlorophenols degradation, a potential remediation for fungicides residues in leather

Journal of Leather Science and Engineering volume 4, Article number: 21 (2022) Cite this article

A Correction to this article was published on 06 May 2023

This article has been updated

Abstract

Chlorophenols, used as the fungicides in leather, are strictly limited in leather products. In this work, a metal–organic framework material, zeolitic metal azolate framework-7 (MAF-7), was first used to encapsulate laccase (Lac) to prepare MAF-7/Lac bio-composites with 98.5% immobilization yield. Afterward, Lac/MNP@MOM was formed by introducing the magnetic nanoparticles (MNPs) into the Lac@MOM. MAF-7 with better hydrophilicity and stronger pH buffering ability, exhibits good compatibility with laccase, which can reserve the activity of laccase after immobilization. Moreover, the porous structure of MAF-7 is favorable for the sufficient contact between laccase and substrates. Lac/MNP@MOM exhibited excellent activity when exposed to high temperature, extreme pH, and organic solvents, which also simplified complex recovery steps. Furthermore, the degradation rate of 2,4-dichlorophenol (2,4-DCP) could reach as high as 97% within 24 h by immobilized laccase, and after nine consecutive cycles of operation, enzyme activity could remain over 80%, which gives it the potential for practical applications.

Graphical abstract

1 Introduction

Phenols and their derivatives are mainly used as pesticides in industry, such as leather, textile and plastics [1]. However, the residues from the phenolic matrix were identified to threaten the environment and organisms [2, 3], and included as a priority pollutant in the list issued by the United States Environmental Protection Agency (USEPA), requiring that the content of phenol in drinking water must be less than 1 μg/L [4]. Therefore, it is meaningful to exploit efficient methods for removing such low-concentration, highly toxic, and difficult-to-degrade pollutants. At present, physical adsorption [5, 6], chemical oxidation [7, 8], and microbiological involved methods [9, 10] are common pathways applied for the treatment of phenolic pollutants, which may inevitably cause secondary pollution or time–cost [11]. Gradually, the enzyme treatment method emerges as a useful tool for phenolic pollutants treatments, with advantages of mild operating conditions, nontoxic by-products, and excellent degradation efficiency [12].

Laccase, first reported in 1883, belongs to the superfamily of multicopper oxidases (MCOs)-a group of enzymes comprising many proteins with different substrate specificities and diverse biological functions [13]. The presence of cupredoxin-like domains allows laccase to reduce oxygen to water without producing harmful byproducts, accompanied by the oxidation of the substrate, especially phenolic compounds [14]. Laccase oxidizes phenols to active radicals that polymerize to form dimers or polymers with reduced solubility and toxicity in water, allowing easy separation from water, which avoids the problems of secondary pollution and separation [15, 16]. Li et al. [17] described that catechol could be degraded by 88.4% within 2 h in the presence of free laccase; Zhang et al. [18] found that the removal efficiency of 2,4-dichlorophenol (2,4-DCP), 2-chlorophenol (2-CP), and 4-chlorophenol (4-CP) could reach 94%, 75%, and 69% within 10 h on laccase, respectively. In addition, laccase is considered as an efficient enzyme for dye degradation, and the application of laccase to degrade leather dyes enables cleaner production [19,20,21]. However, the environmentally sensitive, low stability, and unrecyclable nature of free laccase have limited its industrial application to a certain extent [22].

Enzyme immobilization is an ideal method to compensate for the deficiency of free enzymes, which contains the selection of suitable supports and reasonable application of the effects of support-enzyme interaction[23, 24]. Metal–organic frameworks (MOFs), with high encapsulation rate [25], porous crystallite structures [26], low cytotoxicity [27], and good biocompatibility [28], have been widely applied in enzyme immobilization [29]. Lin et al. [30] encapsulated laccase into ZIF-90 that was combined with adenosine triphosphate and designed an ultrasensitive online electrochemical detector with a detection limit as low as 0.12 nm; Wei et al. [31] encapsulated laccase into ZIF-8 to prepare a glucose biosensor, which maintained good stability after 15 h of continuous work. Among immobilized materials, MAF-7 has better hydrophilicity and stronger pH buffering ability [32], which was more compatible with enzymes and leads to retention of catalytic activity. In addition, the confined structure of MAF-7 could inhibit the undesirable aggregate or conformational change of laccase, and the size of MAF-7 channel is suitable for the transport of substrates and products. Thus, immobilization of laccase on MAF-7 is still desirable although no researchers have encapsulated laccase in MAF-7.

Meanwhile, the introduction of magnetic nanoparticle Fe3O4 into enzyme immobilization system can simplify the operation and separation process, and facilitate the recovery of enzyme[33]. The electron spin state of the intermediate product can be affected and the activation of enzyme can be promoted by magneto-induced effect in the presence of Fe3O4 nanoparticles during the reaction process. The remediation of pyrene polluted soil was reported that achieved by iron nanoparticles and the removal rate of pyrene increased with the increase of iron nanoparticles [34]. Liu et al. [35] immobilized the laccase on Fe3O4@SiO2-NH2 nanoparticles, and the removal rate of 2,4-DCP is still more than 59% after six times of recycling.

In this study, it is the first time that laccase and Fe3O4 were both encapsulated in the MAF-7 by a mild and simple one-step synthetic approach, affording magnetically and catalytically active composite (Scheme 1). After immobilization, the crystalline structure with the dodecahedral rhombus structure of MAF-7 was destroyed, therefore, the material which used to encapsulate laccase was named MOM (Metal Organic Material) in the manuscript. Compared with the reported work and the typical reaction catalyzed by laccase, we found that the immobilized laccase displayed excellent catalytic activity and stability in harsh conditions, and retained a high percentage of its initial activity after a month. Also, the immobilized laccase included the unsurpassed removal rate of 2,4-DCP, and remained surprisingly activity after nine consecutive operations, which provided the possibility to degrade fungicides residues in leather manufacture.

Scheme 1
scheme 1

Schematic diagram for the synthesis of Lac/MNP@MOM

Full size image

2 Experimental section

2.1 Materials

Zinc nitrate hexahydrate (Zn (NO3)2⋅6H2O), 3-methyl-1H-1,2,4-triazole (Hmtz), FeCl3⋅6H2O, FeCl2⋅4H2O, and NH3⋅H2O (25%) were purchased from Sigma Aldrich (Shanghai, China). 2,4-Dichlorophenol (2,4-DCP), 4-aminoantipyrine (4-AP), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and 2-(N-morpholino) ethanesulfonic acid (MES) were obtained from Macklin (Shanghai, China). The laccase from Aspergillus oryzae (Lac) was supplied by SUNSON (Ningxia, China) and stored at 4 °C. A Bradford Protein Assay Kit for protein (0.1–1.5 mg mL−1) was provided by Beyotime (Shanghai, China).

2.2 Synthesis method for magnetic nanoparticles (MNPs)

The magnetic nanoparticles (MNPs) were prepared by previously reported with slight modifications [36]. 80 mL of deionized water (DI water) and magnetic stirrers were placed in a 250 mL double-necked round-bottomed flask, and the oxygen present was purged with nitrogen for half an hour. Subsequently, FeCl3⋅6H2O (27 g, 0.010 mol) and FeCl2⋅4H2O (0.99 g, 0.005 mol) were added and reacted at 50 ℃ for 1 h. Under vigorous stirring, ammonia aqueous solution (10 mL, 25% w/w) was slowly added, and the reaction was continued at 70 ℃ for 3 h. MNPs were obtained by magnetic separation, washed with water for three times, and ethanol for three times respectively, and finally stored at ethanol solution at -4 ℃.

2.3 Synthesis method for Lac/MNP@MOM, Lac@MOM, and MAF-7

The approach was reproduced as described in the literature with some modifications. Laccase and Zn (NO3)2 aqueous solution (1 mL, 0.12 mol L−1) were rapidly mixed with an aqueous solution containing Hmtz (2 mL, 0.18 mol L−1), MNPs, and NH3·H2O (10%). The solution changed from clear to opaque after 24 h of continuous agitation at room temperature. Finally, Lac/MNP@MOM was recovered from the sediment by magnetic separation, and washed with water for three times.

Following the procedure for Lac/MNP@MOM, Lac@MOM was obtained in the absence of MNPs and was gathered by centrifugation at 8000 rpm for 5 min. Pure MAF-7 was prepared by a similar method without MNPs and laccase.

2.4 Characterization

Scanning electron microscopy (SEM) was performed using a Hitachi SU8100 instrument. X-ray diffraction (XRD) data were obtained by Rigaku Smart Laboratory at 9 keV in Bragg–Brentano geometry using Cu κα radiation (λ = 1.5405 Å) as the X-ray source, and the scan speed was 10 min−1, step 0.02°, and 2θ was in the range between 5° and 30°. The spatial distribution of fluorophore-tagged laccase within the MOF composites was determined using the CLSM technique (UP-Sigma, Rtec). Fourier transform infrared (FT-IR) spectra of the samples were recorded on a Bruker ALPHA FT-IR spectrometer (Bruker Vertex 70) using the KBr disk method. The X-ray photoelectron spectroscopy measurement (XPS) was obtained with Thermo Fisher Scientific K-Alpha. Thermal gravimetric analysis (TGA) data were obtained on a simultaneous thermal analyzer (STA) (TGA/DSC) from Linseis Thermal Analysis, then, samples were filled into an alumina crucible and heated in a continuous flow of nitrogen gas from room temperature up to 800 °C. A vibrating sample magnetometer (VSM Lakeshore 7404) was utilized to analyze the magnetic values of the samples.

2.5 Laccase activity assay and steady-state kinetic assay

Following the method in previously reported literature [37], the activity of laccase was determined by using ABTS as the substrate. Generally, the reaction mixture containing a suitable amount of laccase (free laccase or immobilized laccase) and ABTS (1 mmol L−1) solution prepared by citrate buffer solution (0.1 mol L−1) were reacted for 5 min under the respective optimum temperature conditions. The activity of laccase was measured at 420 nm using a UV–vis spectrophotometer, one unit (1 U) of activity was defined as the amount of laccase required to produce 1 μmol ABTS to green chelate within one minute. The pure MAF-7 sample without laccase was measured, and the result showed that MAF-7 exhibited no catalytic activity. All activities were measured three times in parallel, and the results were averaged.

The relative enzyme activity was calculated by the ratio between the measured enzyme activity and the initial enzyme activity (100%), as shown in the following equation:

$${\text{Relative}}\;{\text{activity}}(\% ) = \frac{{{\text{measured}}\;{\text{enzyme}}\;{\text{activity}}}}{{{\text{initial}}\;{\text{enzyme}}\;{\text{activity}}}} \times 100\%$$

The Michaelis–Menten kinetic parameters (Km and Vmax) were obtained by measuring the initial reaction rate with ABTS at 420 nm. The reactions were carried out at 30 ℃ in citrate buffer solution (pH 3.5), and the concentration of ABTS ranged from 0.1–3 mmol L−1. The Lineweaver–Burk equation was employed to calculate Km and Vmax.

$$\frac{1}{V} = \frac{{K_{{\text{m}}} }}{{V_{\max } \left[ {\text{S}} \right]}} + \frac{1}{{V_{\max } }}$$

[S]—substrate concentration (mM);V—reaction rate (mM·min−1).

2.6 Laccase activity in harsh conditions

Free laccase, Lac@MOM, and Lac/MNP@MOM were incubated at 50 ℃, 60 ℃, 70 ℃, and 80 ℃ for 30 min to explore the thermal stability of laccase. The investigation of pH stability, free laccase, Lac@MOM, and Lac/MNP@MOM were incubated in solutions with different conditions (30 mM MES, pH 3, 4, 7, 9, 10) for 30 min. The polar and nonpolar solvent stability, Lac@MOM and Lac/MNP@MOM were stored in ethyl acetate (EA), dichloromethane (DCM), ethanol (EtOH), dimethyl sulfoxide (DMSO), and N, N-dimethylformamide (DMF) for 30 min. Then, the precipitates were recovered and washed three times with DI water, and the residual activity was analyzed. To investigate the storage stability, the free and immobilized laccases were stored in a refrigerator at 4 ℃ for 30 days, and the enzyme activity was measured every 5 days. The initial enzyme activity was defined as 100%.

2.7 Removal of 2,4-dichlorophenol

To investigate the degradation capacity of immobilized laccase towards hazardous phenolic compounds, 2,4-DCP was selected as object to compare the degradation effects of free laccase. The biological removal of 2,4-DCP was performed by adding immobilized enzyme (0.1 mL, 1 mg mL−1) to a phenolic mixture (1 mL) containing 2,4-DCP (0.1 mg mL−1) configured with MES buffer solution (30 mM). The supernatant was obtained by magnetic separation or centrifugation, and the concentration of 2,4-DCP was determined by high-performance liquid chromatography (HPLC) on a C18 reversed-phase column (Inertsil ODS-4, 150 mm × 4.6 mm, 3 μm particles). The detection conditions were as follows: the mobile phase was V (methanol): V(water) = 30: 70, the flow rate of 0.8 mL min−1, and the detection wavelength was 226 nm. Before HPLC analysis, the samples were filtered through a 0.22 µm filter to remove insoluble compounds. The removal efficiency (RE) for 2,4-DCP can be computed by the following equation:

$${\text{RE}}\left( \% \right) = \left( {C_{{\text{o}}} - C_{{\text{t}}} } \right)/C_{0} \times 100\%$$

C0—initial concentration of 2,4-DCP (mg mL−1);Ct—concentration of 2,4-DCP at t h (mg mL−1).

To investigate the effect of pH on the biological removal of 2,4-DCP by free or immobilized laccase, phenol mixture with different pH values (3–10) were used to catalyze the reaction at 30 °C for 24 h. The influence of temperature was studied by evaluating the RE values of 2,4-DCP in the mixture catalyzed by free or immobilized laccase at different temperatures (25–55 °C) for 24 h. Then, the 2,4-DCP mixture was removed by Lac@MOM and Lac/MNP@MOM for 24 h at the respective optimal pH and temperature, and the RE values were measured. The reusability of Lac@MOM and Lac/MNP@MOM under optimal reaction conditions were evaluated by continuously and repeatedly removing phenolic mixtures. After each cycle, the samples were washed with deionized water three times and then added to the next reaction, initial laccase activity was defined as 100%. Moreover, the influence of substrate concentration was tested for Lac/MNP@MOM under optimal reaction conditions.

3 Results and discussion

3.1 Characterization of as-prepared composites

MAF-7, Lac@MOM, and Lac/MNP@MOM composites were investigated by SEM. As shown in Fig. 1a-c, the appearance of MAF-7 was to be a dodecahedral rhombus structure with a diameter of 2 μm despite their rough edges, which is consistent with that reported in literature [38]. While the morphology of Lac@MOM and Lac/MNP@MOM composites became more spherical, the particle sizes were unevenly distributed after the enzyme was immobilized, possibly due to laccase-mediated aggregation growth dynamics [39,40,41]. Additionally, XRD patterns were performed for MAF-7, Lac@MOM, and Lac/MNP@MOM (Fig. S1). Bare MAF-7 exhibited diffraction peaks at 2θ values of 7.34°, 10.38°, 12,72°, 14.72°, 16.46°, and 18.04°, which is consistent with the previously published data for MAF-7 crystal, confirming that MAF-7 had a pure sodalite topology. Compared with MAF-7, the peak intensities of Lac@MOM and Lac/MNP@MOM significantly decreased, as the site of MAF-7 is occupied by enzyme molecules, thereby destroying the integrity of its crystal structure.

Fig. 1
figure 1

SEM images of MAF-7 a, Lac@MOM b, and Lac/MNP@MOM c; Confocal laser scanning micrographs showing the fluorescence d, bright field e, and overlay f images of Lac/MNP@MOM

Full size image

Furthermore, to inquire the location of laccase in Lac/MNP@MOM composites and ensure the successful incorporation of laccase into MAF-7, the fluorescent FLac/MNP@MOM was synthesized by applying FLac as the reactant for CLSM imaging. The confocal microscopy of FLac@MOM revealed a good colocalization of laccase (green fluorescence) and MAF-7 (Fig. 1d-f), indicating laccase is well dispersed in the backbone of MAF-7. Additionally, samples of the as-synthesized composites were also characterized by FI–IR as shown in Fig. 2a. As for MAF-7, the peak at 421 cm−1 may be due to Zn-N stretching. Lac@MOM and Lac/MNP@MOM showed two additional peaks at ~ 1544 cm−1 and 1654 cm−1, which was attributed to amide II and amide I bands, respectively [42], implying the successful encapsulation of laccase in MAF-7. Moreover, the peak at 613 cm−1 for the MNP demonstrated the presence of Fe–O bonds, while the peak could not be seen in the MNP@MOM and Lac/MNP@MOM spectrums, it may be considered that MNPs were located inside of MAF-7 frame. Then, the XPS analyses (Figure. S2, Table. S1) showed that iron was evenly distributed in the immobilized enzyme system, which also indicated that the protein molecules of the enzyme were well coated in MAF-7. Meanwhile, the TGA analysis (Fig. 2b) confirmed the presence of laccase and MNPs in the composite material.

Fig. 2
figure 2

a FT-IR spectra of MAF-7, Lac@MOM, Lac/MNP@MOM, and MNP@MOM; b TGA curves of MAF-7, Lac@MOM, and Lac/MNP@MOM

Full size image

The magnetization of MNP@MOM and Lac/MNP@MOM were investigated by obtaining VSM measurements. The magnetization of MNP@MOM was approximately 0.99 emu/g, while the curve at Lac/MNP@MOM show a magnetization saturation value of 2.02 emu/g. Compared with MNPs, the curves of MNP@MOM and Lac/MNP@MOM exhibited a decrease in magnetization saturation (Ms) values (Fig. 3). It could be reasoned that MAF-7 encapsulation results in a decrease in the surface moment of individual MNPs, eventually resulting in a decrease in the net magnetism [43]. Although the surface moments of MNP@MOM and Lac/MNP@MOM were slightly decreased, they still exhibit excellent superparamagnetic properties and can be quickly separated from the liquid in the presence of an external magnetic field.

Fig. 3
figure 3

VSM spectra of MNP (a), MNP@MOM and Lac/MNP@MOM (b)

Full size image

3.2 Catalytic activity and Michaelis–Menten kinetic parameters of Lac@MOM, and Lac/MNP@MOM.

The activities of free laccase, Lac@MOM, and Lac/MNP@MOM were calculated to be 2542.2, 2694.7, and 2426.3 U g−1 protein (Additional file 1: Figure S3), indicating the encapsulation method has no significant effect on enzyme activity. As is shown in Table 1, the Km, and Vmax values of free laccase were 0.119 mM and 0.27 mM min−1 respectively. However, the Km values for Lac@MOM and Lac/MNP@MOM were decreased to 0.080, 0.091 mM, respectively, which was probably caused by the triazole linkers of MAF-7 increasing the affinity between enzymes and substrates, thereby greatly increasing the reaction rate. The Vmax values of Lac@MOM (0.25 mM min−1), and Lac/MNP@MOM (0.23 mM min−1) were lower than that of free laccase, especially the latter, the phenomenon may be caused by the mass transfer limitation for Lac/MNP@MOM composite tends to aggregate in the reaction. Besides, the kcat value of free laccase was higher than that of the immobilized enzyme, the conversion rate of free enzyme is faster, which corresponded to the Vmax value expressed. Consequently, contrast to free laccase, the enhanced kcat/Km for Lac@MOM, Lac/MNP@MOM indicated that immobilization of laccase on MAF-7 could have better catalytic efficiency.

Table 1 Kinetic parameters of free laccase, Lac@MOM, and Lac/MNP@MOM
Full size table

3.3 Laccase activity in harsh conditions

3.3.1 Thermal deactivation studies

It can be seen that the activity of free enzyme, Lac@MOM and Lac/MNP@MOM gradually decreased with the increase of temperature, and the decrease rate of immobilized enzyme was much lower than that of free laccase (Fig. 4a, Additional file 1: Fig. S4). When the temperature increased to 80 °C, the remaining activity of the free enzyme was only 8.8%. However, the remaining activities of Lac@MOM and Lac/MNP@MOM were 42.74% and 38.30%, respectively. This is because MAF-7 can form an “armor” on the surface of laccase, preventing laccase from direct contact with high temperature environment, thereby avoiding the reduction of laccase activity caused by high temperature.

Fig. 4
figure 4

a Thermal inactivation of free laccase (red), Lac@MOM (blue), Lac/MNP@MOM (green) at 50 ℃, 60 ℃, 70 ℃ and 80 ℃ for 30 min; b pH condition deactivation of free laccase (red), Lac@MOM (blue), Lac/MNP@MOM (green) at different pH conditions for 30 min; c Organic solvent stability evaluation of Lac@MOM (blue), Lac/MNP@MOM (green); d Storage stability of Lac@MOM (blue) and Lac/MNP@MOM (green) (4 °C)

Full size image

3.3.2 pH condition deactivation analysis

Enzymes are very sensitive to the pH of the processing system, and adverse conditions usually cause enzyme inactivation. As shown in Fig. 4b, both the free enzyme and the immobilized laccase maintained more than 70% activity under pH 7 (Additional file 1: Fig. S5). Notably, when the pH reached 9, the free laccase activity is reduced to 21.75%, while immobilized enzyme activity still retains more than 80%. Compared with free laccase, Lac@MOM and Lac/MNP@MOM showed higher tolerance to extreme pH and can function normally in a wider pH range. Moreover, MAF-7 affords a robust coating that offers protection, and its confined three-dimensional structure reduces enzymes undesirable aggregate or conformational changes in the basic solution to remain the enzyme activity, So, the immobilized laccase has stronger vitality than that of free laccase under alkaline conditions, which is consistent with the situation reported in the previous literature.

3.3.3 Organic solvent stability studies

Laccase has been used to repair the polluted environment by biological transformation, and the majority of substrates are soluble in organic solvents, which may significantly deactivate enzymes. Thus, enzyme-catalyzed reactions have been evaluated in a variety of polar and nonpolar solvents (Fig. 4c), including ethyl acetate (EA), dichloromethane (DCM), ethanol (EtOH), dimethyl sulfoxide (DMSO), and N, N-dimethylformamide (DMF). After treatment, Lac@MOM and Lac/MNP@MOM still showed excellent catalytic activity (> 79%), the reason interpreted that MAF-7 has an Å-sized pore size, which prevents organic solvents from contacting the embedded enzyme. In addition, compared with Lac@MOM, Lac/MNP@MOM was more affected by organic solvents, which may be because MNPs change the pore size of MAF-7, thereby the probability of contacting organic solvents increases. Since the addition of MNPs involves recycling to a greater extent, the slight difference could be ignored.

3.3.4 Storage stability studies

As shown in Fig. 4d, due to the protective effect of MOFs on the active conformation of laccase, Lac@MOM and Lac/MNP@MOM had 78.7% and 74.1% residual activities after storage for 30 days at 4 °C. Compared with the reported literature, in which the laccase activity decreased significantly after prolonged storage(less than 20%) [44], the immobilized laccase in this work had higher storage stability. The increase in a residual activity fully implies the advantage of the metal–organic framework in further stabilizing the protein structure.

The above results showed that Lac@MOM and Lac/MNP@MOM were much more resistant to high temperature, alkaline pH and organic solvents compared to free laccase, and remained amazing activities after a month storage at 4 °C, indicating that the MAF-7 framework with high stability and special pore structure can effectively protect the activity of laccase from adverse environmental effects, thus providing the necessary stability for practical laccase applications.

3.4 Removal of 2,4 -dichlorophenol

It can be judged that the optimal pH of Lac@MOM and Lac/MNP@MOM for 2,4-DCP degradation is 4 and 5, respectively, and the temperature is 45 ℃ (Fig. S6-S7). Then, the degradation of 2,4-DCP in 24 h was studied by using the Lac@MOM and Lac/MNP@MOM under optimal reaction systems (Fig. 5a, Additional file 1: Fig. S8). After eight hours of reaction, the removal rate of Lac@MOM was 83.25%, which was slightly higher than that of Lac/MNP@MOM (76.68%), it may be caused by Lac@MOM having a better Michaelis constant and maximum reaction rate than that of Lac/MNP@MOM. After 12 h of reaction, the reaction rate of Lac/MNP@MOM and Lac@MOM slowed down, which may be reasoned by the formation of polymers blocked the pores in MAF-7 during the degradation of 2,4-DCP, which reduced the contact area with the substrate or prolonged the reaction and led to enzyme leakage. In short, compared with other immobilized laccase reported in the literature [44,45,46], Lac@MOM and Lac/MNP@MOM exhibit higher catalytic efficiency, due to the better compatibility of MAF-7 and laccase. Additionally, reusability is an important feature of immobilized biocatalysts and reduces the cost of industrial operations. Thus, the reusability of Lac@MOM and Lac/MNP@MOM were investigated to evaluate their performance in downgrading 2,4-DCP under optimal reaction conditions (Fig. 5b). The residual activity remained above 80% after 9 cycles of continuous reuse, and the reduced activity may be attributed to the production of polymers that were bound to laccase or continuously adsorbed on the MOFs shell, or the leakage of enzyme molecules during use. Anyway, the immobilized laccase exhibits potential for industrial applications with a simple recovery process. Moreover, the degradation efficiency decreased with the concentration of 2, 4-DCP increased, however, even at concentration of 0.6 mg/mL, the degradation efficiency of 2, 4-DCP was still 58% (Additional file 1: Table S2).

Fig. 5
figure 5

a Removal efficiency of 2,4-DCP over 24 h; b Reusability of Lac@MOM (blue) and Lac/MNP@MOM (green)

Full size image

4 Conclusion

A one-step process was developed to encapsulate laccase and MNPs into MAF-7 to form Lac @MOM and Lac/MNP@MOM, which eliminates the complex synthesis process, avoids the low load rate hindrance, and enables the recyclability of laccase. The morphology and crystal structure of the immobilized laccase were determined through a series of characterization methods, such as XRD, SEM, and FT-IR. Compared with free enzymes, the synthesized magnetic framework composite has high catalytic efficiency and recyclability and would be more stable in high temperature, extreme pH, and polar solvent environments. In conclusion, Lac/MNP@MOM showed high degradation activity and excellent reusability, meanwhile, apparently reducing the processing costs, which might be expected to be used in industry (e.g. leather manufacture and textile production).

Availability of data and materials

All data generated or analyzed during this study are included in this published article [and its supplementary information files].

Change history

Abbreviations

2-CP:

2-Chlorophenol

4-CP:

4-Chlorophenol

2,4-DCP:

2,4-Dichlorophenol

CLSM:

Confocal laser scanning micrographs

FT-IR:

Fourier transform infrared

Lac:

Laccase

MAF-7:

Zeolitic metal azolate framework-7

MCOs:

Multicopper oxidases

MNPs:

Magnetic nanoparticles

MOFs:

Metal–organic frameworks

Ms:

Magnetization saturation

RE:

Removal efficiency

SEM:

Scanning electron microscopy

TGA:

Thermal gravimetric analysis

USEPA:

The United States Environmental Protection Agency

XPS:

The X-ray photoelectron spectroscopy measurement

XRD:

X-ray diffraction

References

  1. Albuquerque BR, Heleno SA, Oliveira M, Barros L, Ferreira I. Phenolic compounds: current industrial applications, limitations and future challenges. Food Funct. 2021;12:14–29.

    Article  CAS  Google Scholar 

  2. Lin SH, Juang RS. Adsorption of phenol and its derivatives from water using synthetic resins and low-cost natural adsorbents: a review. J Environ Manage. 2009;90:1336–49.

    Article  CAS  Google Scholar 

  3. Bhatnagar A, Sillanpaa M. Applications of chitin- and chitosan-derivatives for the detoxification of water and wastewater–a short review. Adv Colloid Interface Sci. 2009;152:26–38.

    Article  CAS  Google Scholar 

  4. Doğan T, Bayram E, Uzun L, Şenel S, Denizli A. Trametes versicolorlaccase immobilized poly(glycidyl methacrylate) based cryogels for phenol degradation from aqueous media. J Appl Polym Sci. 2015;132:41981.

    Article  Google Scholar 

  5. Wang H, Zhang H, Jiang JQ, Ma X. Adsorption of bisphenol A onto cationic-modified zeolite. Desalin Water Treat. 2016;57:26299–306.

    Article  CAS  Google Scholar 

  6. Chen M, Zhang Z, Zhu L, Wang N, Tang H. Bisulfite-induced drastic enhancement of bisphenol A degradation in Fe3+-H2O2 Fenton system. Chem Eng J. 2019;361:1190–7.

    Article  CAS  Google Scholar 

  7. Lim M, Son Y, Khim J. The effects of hydrogen peroxide on the sonochemical degradation of phenol and bisphenol A. Ultrason Sonochem. 2014;21:1976–81.

    Article  CAS  Google Scholar 

  8. Kang JH, Kondo F. Bisphenol a degradation by bacteria isolated from river water. Arch Environ Contam Toxicol. 2002;43:265–9.

    Article  CAS  Google Scholar 

  9. Huang C, Xu P, Zeng G, Huang D, Lai C, Cheng M, Deng L, Zhang C, Wan J, Liu L. The rapid degradation of bisphenol A induced by the response of indigenous bacterial communities in sediment. Appl Microbiol Biotechnol. 2017;101:3919–28.

    Article  CAS  Google Scholar 

  10. Kampmann M, Boll S, Kossuch J, Bielecki J, Uhl S, Kleiner B, Wichmann R. Efficient immobilization of mushroom tyrosinase utilizing whole cells from Agaricus bisporus and its application for degradation of bisphenol A. Water Res. 2014;57:295–303.

    Article  CAS  Google Scholar 

  11. Kim KH, Ihm SK. Heterogeneous catalytic wet air oxidation of refractory organic pollutants in industrial wastewaters: a review. J Hazard Mater. 2011;186:16–34.

    Article  CAS  Google Scholar 

  12. Bettin F, Cousseau F, Martins K, Boff NA, Zaccaria S, Moura da Silveira M, Pinheiro Dillon AJ. Phenol removal by laccases and other phenol oxidases of Pleurotus sajor-caju PS-2001 in submerged cultivations and aqueous mixtures. J Environ Manage. 2019;236:581–90.

    Article  CAS  Google Scholar 

  13. Janusz G, Pawlik A, Swiderska-Burek U, Polak J, Sulej J, Jarosz-Wilkolazka A, Paszczynski A. Laccase properties, physiological functions, and evolution. Int J Mol Sci. 2020;21:966.

    Article  CAS  Google Scholar 

  14. Baldrian P. Fungal laccases-occurrence and properties. FEMS Microbiol Rev. 2006;30:215–42.

    Article  CAS  Google Scholar 

  15. Guan ZB, Luo Q, Wang HR, Chen Y, Liao XR. Bacterial laccases: promising biological green tools for industrial applications. Cell Mol Life Sci. 2018;75:3569–92.

    Article  CAS  Google Scholar 

  16. Su J, Fu 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.

    Article  CAS  Google Scholar 

  17. Li D, Cheng Y, Zuo H, Zhang W, Pan G, Fu Y, Wei Q. Dual-functional biocatalytic membrane containing laccase-embedded metal-organic frameworks for detection and degradation of phenolic pollutant. J Colloid Interface Sci. 2021;603:771–82.

    Article  CAS  Google Scholar 

  18. Zhang J, Liu X, Xu Z, Chen H, Yang Y. Degradation of chlorophenols catalyzed by laccase. Int Biodeter Biodegr. 2008;61:351–6.

    Article  CAS  Google Scholar 

  19. Mani P, Keshavarz T, Chandra TS, Kyazze G. Decolourisation of acid orange 7 in a microbial fuel cell with a laccase-based biocathode: influence of mitigating pH changes in the cathode chamber. Enzyme Microb Technol. 2017;96:170–6.

    Article  CAS  Google Scholar 

  20. Gong J, Wang F, Ren Y, Li Z, Zhang J, Li Q. Preparation of biomass pigments and dyeing based on bioconversion. J Clean Prod. 2018;182:301–12.

    Article  CAS  Google Scholar 

  21. Pandi A, Marichetti Kuppuswami G, Numbi Ramudu K, Palanivel S. A sustainable approach for degradation of leather dyes by a new fungal laccase. J Clean Prod. 2019;211:590–7.

    Article  CAS  Google Scholar 

  22. Ren D, Jiang S, Fu L, Wang Z, Zhang S, Zhang X, Gong X, Chen W. Laccase immobilized on amino-functionalized magnetic Fe3O4-SiO2 core-shell material for 2,4-dichlorophenol removal. Environ Technol. 2021;2021:1–22.

    CAS  Google Scholar 

  23. Dong Z, Liu Z, Shi J, Tang H, Xiang X, Huang F, Zheng M. Carbon nanoparticle-stabilized pickering emulsion as a sustainable and high-performance interfacial catalysis platform for enzymatic esterification/transesterification. ACS Sustain Chem Eng. 2019;7:7619–29.

    Article  CAS  Google Scholar 

  24. Huang WC, Wei W, Xue CH, Mao XZ. Effective enzyme immobilization onto a magnetic chitin nanofiber composite. ACS Sustain Chem Eng. 2018;6:8118–24.

    Article  CAS  Google Scholar 

  25. Simon-Yarza T, Mielcarek A, Couvreur P, Serre C. Nanoparticles of metal-organic frameworks: on the road to in vivo efficacy in biomedicine. Adv Mater. 2018;30:e1707365.

    Article  Google Scholar 

  26. Liang K, Ricco R, Doherty CM, Styles MJ, Bell S, Kirby N, Mudie S, Haylock D, Hill AJ, Doonan CJ, Falcaro P. Biomimetic mineralization of metal-organic frameworks as protective coatings for biomacromolecules. Nat Commun. 2015;6:7240.

    Article  CAS  Google Scholar 

  27. Lian X, Fang Y, Joseph E, Wang Q, Li J, Banerjee S, Lollar C, Wang X, Zhou HC. Enzyme-MOF (metal-organic framework) composites. Chem Soc Rev. 2017;46:3386–401.

    Article  CAS  Google Scholar 

  28. Ji Y, Wu Z, Zhang P, Qiao M, Hu Y, Shen BX, Li BZ, Zhang X. Enzyme-functionalized magnetic framework composite fabricated by one-pot encapsulation of lipase and Fe3O4 nanoparticle into metal-organic framework. Biochem Eng J. 2021;169:107962.

    Article  CAS  Google Scholar 

  29. Doonan C, Riccò R, Liang K, Bradshaw D. Falcaro1 P, Metal-organic frameworks at the bio-interface: synthetic strategies and applications. Accounts Chem Res. 2017;50:1423–32.

    Article  CAS  Google Scholar 

  30. Zhang W, Wang C, Peng M, Ren G, Li K, Lin Y. ATP-responsive laccase@ZIF-90 as a signal amplification platform to achieve indirect highly sensitive online detection of ATP in rat brain. Chem Commun. 2020;56:6436–9.

    Article  CAS  Google Scholar 

  31. Li X, Feng Q, Lu K, Huang J, Zhang Y, Hou Y, Qiao H, Li D, Wei Q. Encapsulating enzyme into metal-organic framework during in-situ growth on cellulose acetate nanofibers as self-powered glucose biosensor. Biosens Bioelectron. 2021;171:112690.

    Article  CAS  Google Scholar 

  32. Tian Y, Zhou Y, Zong Y, Li J, Yang N, Zhang M, Guo Z, Song H. Construction of functionally compartmental inorganic photocatalyst-enzyme system via imitating chloroplast for efficient photoreduction of CO2 to formic acid. ACS Appl Mater Interfaces. 2020;12:34795–805.

    Article  CAS  Google Scholar 

  33. Zhang T, Zhang X, Yan X, Kong L, Zhang G, Liu H, Qiu J, Yeung KL. Synthesis of Fe3O4@ZIF-8 magnetic core-shell microspheres and their potential application in a capillary microreactor. Chem Eng J. 2013;228:398–404.

    Article  CAS  Google Scholar 

  34. Chang MC, Shu HY, Hsieh WP, Wang MC. Remediation of soil contaminated with pyrene using ground nanoscale zero-valent iron. J Air Waste Manag Assoc. 2007;57:221–7.

    Article  Google Scholar 

  35. Huan W, Yang Y, Wu B, Yuan H, Zhang Y, Liu X. Degradation of 2,4-DCP by the immobilized laccase on the carrier of Fe3O4@SiO2-NH2. Chinese J Chem. 2012;30:2849–60.

    Article  CAS  Google Scholar 

  36. Schejn A, Mazet T, Falk V, Balan L, Aranda L, Medjahdi G, Schneider R. Fe3O4@ZIF-8: magnetically recoverable catalysts by loading Fe3O4 nanoparticles inside a zinc imidazolate framework. Dalton Trans. 2015;44:10136–40.

    Article  CAS  Google Scholar 

  37. Yang CH, Wang MX, Haider H, Yang JH, Sun JY, Chen YM, Zhou J, Suo Z. Strengthening alginate/polyacrylamide hydrogels using various multivalent cations. ACS Appl Mater Inter. 2013;5:10418–22.

    Article  CAS  Google Scholar 

  38. Wang L, Liu G, Ren Y, Feng Y, Zhao X, Zhu Y, Chen M, Zhu F, Liu Q, Chen X. Integrating target-triggered aptamer-capped HRP@metal-organic frameworks with a colorimeter readout for on-site sensitive detection of antibiotics. Anal Chem. 2020;92:14259–66.

    Article  CAS  Google Scholar 

  39. Yimamumaimaiti T, Lu X, Zhang JR, Wang L, Zhu JJ. Efficient blood-toleration enzymatic biofuel cell via in situ protection of an enzyme catalyst. ACS Appl Mater Inter. 2020;12:41429–36.

    Article  Google Scholar 

  40. Li X, He Y, Yang L, He Z, Zhu JJ. Gene/drug-embedded nanoscale metal azolate framework-7 for the reversal of P-glycoprotein-mediated multidrug resistance. Chem Commun. 2021;57:6776–9.

    Article  CAS  Google Scholar 

  41. Velasquez-Hernandez MJ, Astria E, Winkler S, Liang W, Wiltsche H, Poddar A, Shukla R, Prestwich G, Paderi J, Salcedo-Abraira P, Amenitsch H, Horcajada P, Doonan CJ, Falcaro P. Modulation of metal-azolate frameworks for the tunable release of encapsulated glycosaminoglycans. Chem Sci. 2020;11:10835–43.

    Article  CAS  Google Scholar 

  42. Sun Q, Aguila B, Lan PC, Ma S. Tuning pore heterogeneity in covalent organic frameworks for enhanced enzyme accessibility and resistance against denaturants. Adv Mater. 2019;31:e1900008.

    Article  Google Scholar 

  43. Vibhute SP, Mhaldar PM, Shejwal RV, Pore DM. Magnetic nanoparticles-supported palladium catalyzed Suzuki-Miyaura cross coupling. Tetrahedron Lett. 2020;61:151594.

    Article  CAS  Google Scholar 

  44. Qiu X, Wang Y, Xue Y, Li W, 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.

    Article  CAS  Google Scholar 

  45. Donati E, Polcaro CM, Ciccioli P, Galli E. The comparative study of a laccase-natural clinoptilolite-based catalyst activity and free laccase activity on model compounds. J Hazard Mater. 2015;289:83–90.

    Article  CAS  Google Scholar 

  46. Qiu X, Qin J, Xu M, Kang L, 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. Colloid Surface B. 2019;179:260–9.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was financially supported by the National Key Research and Development Project (Grant No. 2021YFC2100100).

Funding

Not applicable.

Author information

Author notes

  1. Min Cao and Jie Yu contributed equally to this work

Authors and Affiliations

  1. School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Nanjing, 210023, People’s Republic of China

    Min Cao, Xing Zhang, Yamei Lin & He Huang

  2. School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing, 210009, People’s Republic of China

    Jie Yu & He Huang

Authors
  1. Min Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jie Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xing Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yamei Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. He Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

MC performed the experiments, analyzed the results and revised the manuscript. JY performed the experiments and wrote the initial draft. YL, XZ and HH reviewed and edited the manuscript. All authors read and approved the final manuscript

Corresponding author

Correspondence to He Huang.

Ethics declarations

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1

. Fig. S1. XRD patterns of MAF-7 (black), Lac@MOM (red), and Lac/MNP@MOM (blue). Fig. S2. XPS spectra of MAF-7, Lac@MOM, and Lac/MNP@MOM (a); XPS spectra of Zn 2p for Lac/MNP@MOM (b); XPS spectra of Fe 2p for Lac/MNP@MOM (v); XPS spectra of Fe 2p for Lac/MNP@MOM (d). Table. S1. Surface element ratios of Lac/MNP@MOM. Fig. S3. Effect of laccase concentration on laccase immobilization. Fig. S4. Temperature effect on laccase activity. Fig. S5. pH effect on laccase activity. Fig. S6. Effect of pH on removal of 2,4-DCP. Fig. S7. Effect of temperature on removal of 2,4-DCP. Table. S2. Effect of substrate concentration on removal of 2,4-DCP. Fig. S8. The UV-Vis absorption of composites catalyzed 2,4-DCP.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Cao, M., Yu, J., Zhang, X. et al. Laccase-functionalized magnetic framework composite enabled chlorophenols degradation, a potential remediation for fungicides residues in leather. J Leather Sci Eng 4, 21 (2022). https://doi.org/10.1186/s42825-022-00094-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s42825-022-00094-3

Keywords