Colloids and Surfaces A: Physicochemical and Engineering Aspects 660 (2023) 130820

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Colloids and Surfaces A: Physicochemical and

Engineering Aspects
journal homepage: www.elsevier.com/locate/colsurfa

Sandwich-like heterostructured nanomaterials immobilized laccase for the

degradation of phenolic pollutants and boosted enzyme stability
Mengyu Li a, Yahan Bai a, Wei Zhuang a, b, *, Jinle Liu a, Zhi Wang a, Yuan Rao a, Mengran Li c,
Hanjie Ying b, Pingkai Ouyang b
a
School of Chemical Engineering, Zhengzhou University, Zhengzhou 450001, China
b
College of Biotechnology and Pharmaceutical Engineering, State Key Laboratory of Materials-Oriented Chemical Engineering, National Engineering Technique Research
Center for Biotechnology, Nanjing Tech University, No. 30, Puzhu South Road, Nanjing 211816, China
c
Materials for Energy Conversion and Storage (MECS), Department of Chemical Engineering, the Delft University of Technology, van der Maasweg 9, Delft 2629 HZ, the
Netherlands

G R A P H I C A L A B S T R A C T

A R T I C L E I N F O A B S T R A C T

Keywords: A novel magnetic 2D/2D heterogeneous structure MXene@NiFe-LDH@Fe3O4 was prepared for immobilization of
2D nanomaterials laccase. In this work, two-dimensional MXene nanosheets with abundant surface functional groups were het­
Heterogeneous assembly erogeneously assembled with layered double hydroxide (LDH) by in situ co-precipitation method, and magnetic
Immobilized laccase
nanoparticle Fe3O4 with excellent biocompatibility and rapid separation of materials and substrates was intro­
Simulated industrial wastewater
duced subsequently, and then silane coupling agent was coated on the surface of MXene@NiFe-LDH@Fe3O4. The
functionalized MXene@NiFe-LDH@Fe3O4 was employed as a carrier to immobilize laccase from Trametes-Ver­
sicolor. The enzyme loading of the nanocomposite material is as high as 167.9 mg/g. Compared with free en­
zymes, the immobilized laccase showed a notable improvement in stability in a wider range of pHs (2.0–8.0),
temperatures (25–60 ◦ C), and organic solvent concentration (1–5 M). The reusability study suggested that after 7
cycles of repeated catalysis, the degradation efficiency could reach 55.5% for 2,4-dichlorophenol, 92.1% for
bisphenol A and70.9% for pyrocatechol. The results provide a new carrier preparation strategy for the efficient
immobilization of laccase.

* Corresponding author at: School of Chemical Engineering, Zhengzhou University, Zhengzhou 450001, China.
E-mail address: [email protected] (W. Zhuang).

https://doi.org/10.1016/j.colsurfa.2022.130820
Received 6 September 2022; Received in revised form 1 December 2022; Accepted 17 December 2022
Available online 22 December 2022
0927-7757/© 2022 Elsevier B.V. All rights reserved.
M. Li et al. Colloids and Surfaces A: Physicochemical and Engineering Aspects 660 (2023) 130820

1. Introduction nanosheets while increasing the specific surface area of the material.
Magnetic nanoparticles were introduced to facilitate the rapid separa­
Phenolic compounds are persistent organic pollutants widely exist­ tion of materials and substrates, the synthesized magnetic composite
ing in the water environment, mainly from petrochemical, paper mak­ MXene@NiFe-LDH@Fe3O4 was used as an immobilized enzyme carrier.
ing, pharmaceutical, paint, etc., with strong toxicity, difficult The surface of the composites was modified with (3-Aminopropyl)trie­
degradation, persistence and other characteristics[1]. Without effective thoxysilane (APTES) to increase the loading of laccase, thus improving
removal, they will cause long-term harm to the water environment[2,3]. the overall catalytic efficiency. The immobilization conditions, enzy­
In recent decades, the treatment strategies of organic wastewater are matic properties and applicability of immobilized enzymes in degrading
mainly based on physical methods, advanced oxidation processes, and organic pollutants (2,4-DCP, BPA, pyrocatechol) were also investigated.
biological methods[1,4,5]. Among them, the physical method separates
organic pollutants from wastewater based on physical processes, such as 2. Materials and methods
adsorption, extraction, and precipitation, which only involve the phase
transfer of the pollutants without substantial degradation. The advanced 2.1. Materials
oxidation process has the advantages of strong oxidation capacity and a
wide application range[6]. However, continuous application of energy Laccase (E.C. 1.10.3.2) from Trametes versicolor was purchased from
or chemicals is required during the treatment process[7]. Therefore, the Sigma-Aldrich (Shanghai, China). Sodium dihydngen phoshate anhy­
high energy consumption and cost are inevitable shortcomings. The drous (Na2HPO4), citric acid monohydrate, glutaraldehyde (50%),
biological method has the advantages of environmental protection, low lithium fluoride (LiF), urea, (3-Aminopropyl) trimethoxysilane (APTES),
operating cost and high degradation efficiency, so it has attracted great 2,2′ -azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), 1-Methyl-2-
attention of researchers globally in recent years[4,6,8]. pyrrolidinone (NMP), ferric chloride hexahydrate (FeCl3⋅6 H2O),
Laccase is a single electron oxidoreductase containing copper[9]. In ferrous chloride tetrahydrate (FeCl2⋅4 H2O), ferric nitrate nonahydrate
the presence of oxygen, four copper ions in laccase interact with free (Fe(NO3)3⋅9 H2O), nickel nitrate hexahydrate (Ni(NO3)2⋅6 H2O),
radicals from reactive substrates and synergically transfer electrons NH3⋅H2O (28–30%), sodium hydroxide (NaOH), 2,4-dichlorophenol
through the three-nucleus copper cluster center, thus catalyzing the (2,4-DCP, purity ≥ 98%), bisphenol A (BPA, purity ≥ 99.0%) and py­
oxidation of lignin, phenol, aromatic amine, chlorophenol, thiophenol, rocatechol (purity ≥ 99.5%) were purchased from Aladdin (Shanghai,
dyes, etc.[10,11]. It is a widely used environment-friendly enzyme China). Ti3AlC2 (MAX, 200mesh) was obtained from Macklin (Shanghai,
catalyst[12]. However, water-soluble enzymes suffer from poor stabil­ China). Hydrochloric acid (HCl) was supported by Sinopharm Chemical
ity, susceptibility to inactivation, and difficulty in reuse[13]. Therefore, Reagent Co., Ltd.
enzyme immobilization technology methods such as adsorption[14]、
cross-linking[15] and embedding[16] have been introduced to over­ 2.2. Synthesis of MXene@NiFe-LDH nanohybrid
come these limitations.
The selection of carriers is the key to the immobilization process[17], Ti3C2TX-MXene nanosheets were prepared according to the reported
a series of nanomaterials of different sizes and shapes have been re­ method[24]. Firstly, the LiF (1.8 g) was dispersed in HCl (20 mL) and
ported in the field of biotechnology as new types of immobilized enzyme magnetically stirred for 10 min, Ti3AlC2 (1 g) was gradually added into
carriers[18,19]. MXene is a two-dimensional layered material composed the etching solution and continuously stirred for 24 h at 35 ◦ C. Subse­
of transition metal carbides, nitrides or carbonitrides[20], which can be quently, the resulting suspension was washed with deionized water
prepared by selectively etching the main group element A layer from the several times until the pH of the liquid became neutral. After that, the
corresponding MAX phase (A = Al, Si, Ge and other main group ele­ obtained intermediate product was further dispersed in deionized water,
ments)[21], with large surface area, hydrophilicity, tunable conductiv­ and the mixture was sonicated in ice-water bath for 2 h. Finally, by
ity and abundant surface functional groups (-OH, = O, -F)[22]. centrifuging at 4000 rpm for 30 min, the supernatant was poured to
However, due to Van der Waals forces and interlayer π-π interactions obtain a black Ti3C2TX-MXene colloidal solution with a concentration of
[23], MXene as a carrier exhibits a strong tendency to agglomerate, 5.88 mg/mL.
resulting in stacking of nanosheets, hindering mass transfer and masking The obtained Ti3C2TX-MXene colloidal solution (8.5 mL) was
active sites[24]. dispersed in 30 mL N-methyl-pyrrolidone and sonication for 10 min,
Combining two-dimensional materials with zero-dimensional nano­ followed by the addition of a mixed metal salt precursor consisting of Ni
particles, one-dimensional nanowires or two-dimensional nanosheets to (NO3)2⋅6 H2O (3 mM), Fe(NO3)3⋅9 H2O (1 mM) and urea (12 mM). After
reasonably construct nanocomposite microstructures can effectively stirring for 30 min, the solution was subjected to reflux at 100 ◦ C for 5 h.
improve the stacking problem of nanosheets[25]. Layered Double Hy­ Afterwards, the Ti3C2TX loaded with NiFe-LDH (MXene@NiFe-LDH,
droxides (LDHs) are typical layered materials, which have attracted abbreviated as ML) was harvested and washed with deionized water and
attention from the fields of water environmental treatment and elec­ ethanol for several times before drying in freeze dryer.
trochemical energy storage in recent years due to its unique layered Fe3O4 nanoparticles (IONPs) was in situ co-deposited on the surface
structure and excellent exchangability of metal ions and interlayer an­ of ML. Briefly, the mixed metal salt precursors containing FeCl3⋅6 H2O
ions on the laminates[26,27], its highly tunable 2D interlayer channel, (2 mM) and FeCl2⋅4 H2O (1 mM) were dissolved in 50 mL degassed ul­
can significantly improve the intercalation rate of biomolecules[28]. trapure water, 0.5 g ML powder was added after magnetic stirring for 10
Compared with 2D MXene nanosheets, the 2D/2D heterogeneous min, then the pH of suspension above was adjusted to 10 with NH3⋅H2O,
structure of MXene@LDH provides superior specific surface area, and the reaction was stirred for 1 h under the protection of N2 at 80 ◦ C.
adsorption sites and biocompatibility while improving the self-stacking Afterwards, the product was washed several times with deionized water
of nanosheets. However, the nanomaterials currently used for laccase until the pH value of the liquid was neutral. After freeze-drying, MXe­
immobilization are usually in powder form, which is difficult to recover ne@LDH@Fe3O4 (abbreviated as MLF) powder was obtained.
in practical applications. The deposition of magnetic particles on the
surface of MXene@LDH can impart magnetically responsive properties 2.3. Functionalization of MLF
to the composites and achieve rapid separation from the aqueous phase
under the action of an external magnetic field, making them excellent APTES was used to modify the surface of the composite material.
carriers for enzyme immobilization[29]. Normally, 100 mg of MLF powder is dispersed in 100 mL of 50% ethanol
In this study, we prepared MXene@NiFe-LDH nanocomposites using aqueous solution and sonicated for 30 min. Then add 0.5 mL APTES and
a chemical co-precipitation method to improve the self-stacking of stir at room temperature for 10 min, then stir for 10 h under the

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M. Li et al. Colloids and Surfaces A: Physicochemical and Engineering Aspects 660 (2023) 130820

protection of nitrogen at 65 ◦ C. After the reaction, the precipitate was of reaction system (L); ε is the molar extinction coefficient of ABTS2+
washed with ethanol and deionized water to remove unbound APTES, (36,000 M− 1 cm− 1); t is the reaction time (min); m1 and m2 are the
and the MXene@LDH@Fe3O4-NH2 (abbreviated as MLF-NH2) was amount of MLF-lac (g) and protein content of MLF-lac (g), respectively.
freeze-dried and stored at 4 ◦ C for further use.
2.7. Optimization of immobilization conditions
2.4. Characterization
The MLF-NH2 carrier activated by different mass fractions (0%
The morphology and structure of Ti3C2TX-MXene, ML and MLF were ~3.0%) of glutaraldehyde solution was used for the immobilization of
measured by scanning electron microscopy (SEM, Helios G4 CX) and laccase after reacting in a shaker at 25 ◦ C for 2 h. To test the relative
transmission electron microscopy (TEM, JEM-1400Flash) equipped with activity of the immobilized enzyme, the data with the highest activity
energy dispersive spectrometer (EDS). The thickness of the Ti3C2TX- was defined as 100%, and calculate the enzyme loading.
MXene nanosheets was measured by atomic force microscope (Dmen­ The effects of pH on the activities of MLF-lac were studied in a citric
sion fastscan, Bruker). The automatic specific surface and pore size acid-disodium phosphate buffer with different pH values (2.0–9.0) at
distribution analyzer was used to evaluate the specific surface area of the 25 ◦ C. The relative activity at 40 ◦ C was used as the control (100%) to
sample (BET-Autosorb-iQA3200–4). The Fourier transform infrared calculate and compare the activity of MLF-lac, then calculate the
spectroscopy (FTIR, Perkin Elmer-Spectrum 100) was used to investigate enzyme loading.
the surface functionalities of the samples. The structure and phase The effect of temperature on the activities of MLF-lac was examined
characteristics of the samples were characterized by X-ray diffraction in a citric acid-disodium phosphate buffer at 20–80 ◦ C for 30 min at
(XRD, Bruker D8 Advance). The change of the composition and chemical constant pH of 4.0. The relative activity at pH 4.0 was used as the control
states was characterized by an X-ray photoelectron spectroscopy (XPS, (100%), and the remained activity of laccase was analyzed under the
Thermo ESCALAB-250xi). The distribution of enzyme molecules which standard conditions above, then calculate the enzyme loading.
labeled by fluorescein isothiocyanate (FITC) was investigated using
confocal laser scanning microscopy (CLSM, TCS SP5II, Leica). The 2.8. Stability of free and immobilized laccase
vibrating sample magnetometer (VSM, Quantum-MPMS3) was used to
examine the magnetic properties of the carriers. Ultraviolet-visible To investigate the thermal stability, free and immobilized laccase
spectroscopy system (UV–vis, Agilent Cary 60) was used to test the were cultured in 60 ◦ C water for a period of time and the activity was
peroxidase-like performance of the composite material, the loading measured every hour. The enzymatic activity without incubation was
amount of laccase, and the activity of free and immobilized laccase. The taken as the control (100%) to study the remaining activity (%) of free
Zeta potential analyzer (Mastersizer 3000, Malvern) was used to assess and immobilized laccase.
the number of charges and electrostatic interactions on the nanosheets Organic solvent resistance was determined by incubating free and
surface. immobilized laccase in a series of concentrations of urea solution for
30 min. The activity was measured under the optimal conditions
2.5. Immobilization of laccase on MLF-NH2 mentioned above, and the initial activity was defined as 100%.
Storage stabilities of free and immobilized laccase were assessed by
The MLF-NH2 nanocomposites were first dispersed in citric acid- measuring the remaining activity after being stored at 4 ◦ C for one
dibasic sodium phosphate solution (100 mM, pH 4.0) to form a 5 mg/ month, and the laccase activity was determined every five days. The
mL aqueous suspension. The laccase was also dissolved in a citric acid- laccase activity without incubation was taken as the control (100%), the
dibasic sodium phosphate solution to form a 1 mg/mL buffered laccase residual activity was calculated and compared according to the method
solution. The immobilization was carried out by mixing 1.0 mL MLF- described above.
NH2 disperse solution and desired amount of laccase and then incu­
bating at 4 ◦ C with shaking at 180 rpm. We separated the carrier with a 2.9. Reusability of immobilized laccase
magnet, collected the supernatant and washing liquid, collected the
unreacted laccase. The amount of immobilized laccase was calculated The reusability was measured by evaluating the activity of the
using the Bradford assay method. The MLF-NH2 loaded with laccase immobilized laccase activity after each cycle of removing the phenolic
(MLF-NH2-laccase, abbreviated as MLF-lac) was stored at 4 ◦ C until the mixture (containing 0.5 mM ABTS) using MLF-lac (30 ◦ C, pH 4.0).
using time. Further, the catalyst was washed several times with ultra-pure water
after each cycle. The initial laccase activity was defined as the control
(100%).
2.6. Laccase activity assay

2.10. Removal of phenols

Spectrophotometric determination of laccase activity was carried out
by measuring the oxidation of ABTS rate according to the methods re­
Immobilized laccase or an equal amount of laccase was added to
ported in the previous work[30]. Briefly, the reaction system containing
10 mL of 2,4-DCP (10 mg/L), BPA (10 mg/L) or pyrocatechol (10 mg/L)
an aliquot of free laccase, MLF-lac (5 mg) and ABTS solution (0.5 mM, 3
in an aqueous solution with ABTS (0.5 mM), which serve as a mediator
mL) prepared by citric acid-dibasic sodium phosphate buffer solution
substance[32]. The degradation of phenols took place in a shaker at
(100 mM，pH 4.0) was allowed to react for 3 min at 25 ◦ C. The su­
30 ◦ C and 180 rpm for 12 h, the suspension was filtrated out with a
pernatant was filtered with a 0.22 µm filter, and the absorbance was
0.22 µm filter after degradation. The degradation degree was deter­
measured at 420 nm using a UV-Vis spectrometer. One unit of the ac­
mined based on high-performance liquid chromatography with a C18
tivity (U) was defined as the amount of laccase required to catalyze the
reversed-phase column (Agilent，150 mm × 4.6 mm, 5 µm particles).
oxidation of 1 μmol ABTS within one minute, the immobilization rate is
Detection conditions of 2,4-DCP: mobile phase 70% methanol solution；
calculated as follows[31].
detection wavelength: 290 nm; column temperature：35 ◦ C; flow rate:
/
Expressed activity(U/g biocatalyst) = A × 106 × Vt (ε × t × m1 ) (1) 0.8 mL/min; time: 10 min. Detection conditions of BPA: mobile phase A
(ACN): B (H2O: THF: phosphoric =1:0.01:0.002 (v:v:v))= 1:1；detec­
/ /
Specific activity (U g protein) = A × 106 × Vt (ε × t × m2 ) (2) tion wavelength: 214 nm; column temperature：35 ◦ C; flow rate:
0.8 mL/min; time: 10 min. Detection conditions of pyrocatechol: mobile
Where A is the absorbance of ABTS2+ at 420 nm; Vt is the total volume phase 50% methanol solution；detection wavelength: 280 nm; column

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M. Li et al. Colloids and Surfaces A: Physicochemical and Engineering Aspects 660 (2023) 130820

temperature：35 ◦ C; flow rate: 0.8 mL/min; time: 10 min. The degra­ Fe3O4 with a particle size of about 30 nm in the MLF are attached to the
dation efficiency was calculated using the formula below[33]: ML surface (Fig. 2d). TEM further confirmed the sheet structure of
/ Ti3C2Tx-MXene and the successful assembly of the composite materials
Removal efficiency (%) = (Co − Cf ) Co × 100% (3)
(Fig. 2g, h). The elemental analysis of the nanoparticles using EDS
confirmed the presence and uniform distribution of C, Fe, Ni, Ti and O
Where Co and Cf represent the initial and the residual concentration of
atoms (Fig. 2i-n) in ML.
phenols after the enzyme treatment, respectively.
The N2 adsorption/desorption analysis further demonstrated that the
heterogeneous assembly of Ti3C2TX-MXene with NiFe-LDH can effec­
3. Results and discussion
tively increase the specific surface area of the composites. ML and MLF
show a type IV adsorption isotherm with H3-type hysteresis loop
Fig. 1 illustrates the synthesis route of nanocomposites for laccase
(Fig. 3). The BET specific surface area and pore data are listed in Table 1,
immobilization and its application for organic phenols. Firstly，we
proving the existence of the mesoporous structure. It can be speculated
obtained single few-layer nanosheets Ti3C2TX-MXene by etching the Al
that the longitudinal alignment of NiFe-LDH on the surface of Ti3C2TX-
layers from Ti3AlC2 MAX phase and dispersed it in water. Thus, urea and
MXene is the main source of mesopores, which is proved by the BET and
metal salts were added to the colloidal solution, urea hydrolysis caused
pore volume data. After loading NiFe-LDH on the surface of Ti3C2TX-
the precipitation and crystallization of metal salts to achieve anisotropic
MXene, we noted that the specific surface area increased from 1.8 m2/g
growth of NiFe-LDH on the surface of MXene nanosheets, which
to 123.3 m2/g, and the pore volume also increased by about 40 folds.
significantly improve the specific surface area. We then deposited Fe3O4
Although the assembly of magnetic nanoparticles decreased the specific
nanoparticles on the surface of the synthesized nanocomposite to confer
surface area of ML to 114.3 m2/g, it can still be demonstrated that the
magnetic properties and facilitate separation. APTES then modified the
NiFe-LDH can creat more abundant active sites.
surface of the composites by hydrolysis reaction, and the functional
The crystal structures of all nanomaterials were examined by XRD.
group of glutaraldehyde covalently attached the enzyme molecule to the
As shown in Fig. 4a, the (002) at the center position of 7.0◦ is a char­
surface of the carrier for the immobilization of the enzyme.
acteristic peak of Ti3C2TX-MXene, the diffraction peaks including (003),
(006) and (009) are the characteristic peaks of NiFe-LDH crystal struc­
3.1. Synthesis and characterization ture, in line with previous reports[34,35]. In the nanocomposite ML, the
(002) characteristic peak from Ti3C2TX-MXene can be clearly found, but
The nanomaterials from each synthesis stage are monitored by the the characteristic XRD peak position is shifted from the original 7◦ to a
scanning electron microscope and transmission electron microscope lower angle at 5.6◦ , it is indicated that the doping of NiFe-LDH increases
(Fig. 2). The exfoliated Ti3C2Tx-MXene nanosheets show a typical two- the interlayer spacing between Ti3C2TX-MXene nanosheets. XRD results
dimensional layered structure with a lateral size of 5–8 µm (Fig. 2a). confirm the successful combination of two materials. The characteristic
The bending and wrinkles of the nanosheets indicate a high degree of peaks of NiFe-LDH are discernable in the MLF, but the (002) diffraction
flexibility. The AFM image shows that the peeled nanosheets have a flat peak of Ti3C2Tx-MXene is weak and overlapped. MLF had the same
surface and a thickness of about 2.5 nm (Fig. S1). Pure LDH behaves as a crystalline characteristic peaks of the ML, which is an indication that the
nanosphere structure with a diameter size of 1.5–2 µm (Fig. 2b). The ML crystal structure was completely maintained. However, the intensity of
nanohybrid shows a 2D/2D heterostructure, where NiFe-LDH grew in the MLF diffraction peaks is weak, indicating a low degree of
situ and was evenly distributed at the surface of a single layer of Ti3C2Tx- crystallization.
MXene (Fig. 2c). Obviously, ML provides a larger substrate contact area The FTIR spectra of MLF and MLF-NH2 are shown in Fig. 4b. The
and more active sites than the Ti3C2Tx-MXene, which could benefit the absorption peak at 3350 cm− 1 is attributed to the stretching vibration of
charge transfer during the catalytic reaction. Magnetic nanoparticles

Fig. 1. Schematic graphic of the preparation of the immobilized laccase and degradation of organic phenols.

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M. Li et al. Colloids and Surfaces A: Physicochemical and Engineering Aspects 660 (2023) 130820

Fig. 2. SEM images of (a) Ti3C2Tx-MXene, (b) pure NiFe-LDH, (c) ML and (d) MLF nanomaterials; TEM images of (e) Ti3C2Tx-MXene, (f) pure NiFe-LDH, (g) ML and
(h) MLF nanomaterials; (i) TEM dark field scanning and corresponding element mapping showing the uniform distribution of (j) C, (k) Fe, (l) Ni, (m) Ti, (n) O
elements in the ML.

hydrophilic O-H. The peaks at 2973 cm− 1 and 1193 cm− 1 correspond to
the stretching vibrations of C-F and C-O peaks, respectively. This data
confirms the presence of rich functional groups on the MLF surface,
consistent with the previous report[34]. In the spectral comparison of
MLF and MLF-NH2, the additional peaks in the spectrum of MLF-NH2
unveil a strong absorption of Si-O (at 1066 cm− 1)、N-H (at 1634 cm− 1)
and C-N（at 1258 cm− 1）[36]. These peaks directly prove the success­
ful modification of the silane coupling agent at the surface of the com­
posite material.
The XPS survey spectra in Fig. 5a show that the main constituent
elements of MLF nanocomposites are Ni, Fe, Ti, C and O. The presence of
Si and N elements in the sample after surface functionalization with
amino groups indicates that the silane coupling agent was successfully
combined with the carrier. The Ti 2p high-resolution spectrum can be
deconvoluted into four pairs of 2p3/2 and 2p1/2 doublets for Ti-C
(453.7/461.0 eV), Ti2+ (467.2/462.8 eV), Ti3+ (458.8/464.1 eV) and
Ti-O (459.3/456.1 eV) (Fig. 5b)[24], which are the Ti states in the
Ti3C2Tx-MXene nanosheets. The Ni 2p spectrum shows two peaks of 2p
3/2 and 2p 1/2 and two satellite peaks at 861.77 eV and 879.76 eV
(denoted as Sat.) (Fig. 5c)[37]. Two distinct peaks in the Fe 2p spectrum
Fig. 3. N2 adsorption-desorption isotherms of Ti3C2Tx-MXene, NiFe-LDH, ML can be assigned to Fe 2p3/2 (711.1/713.2 eV) and Fe 2p1/2
and MLF nanocomposites. (723.6/726.1) of Fe in Fe3O4 (Fig. 5d)[38]. The peaks of Si 2p can be
associated with Si-O-C (101.70 eV) and Si-O-Si (102.89 eV) (Fig. 5e)
[39], which further confirm the successful of amino-silane modification.
Table 1 The modified amount of silane coupling agent and the relative mass
Pore characteristics for Ti3C2Tx-MXene, NiFe-LDH, ML and MLF. of enzyme molecules on the surface of MLF composites can be estimated
Materials BET VBJH/ (cm3/g) DBJH/ (nm) by thermal gravimetric analysis of MLF, MLF-NH2, and MLF-Lac under
Surface programmed temperature conditions (Fig. 6a). The thermogravimetric
Adsorption Desorption Adsorption Desorption
Area
loss curves of the three materials are shown in Fig. 6b. The MLF lost
（m2/g）
about 21.9 wt% mass during the whole heating process (15–800 ◦ C).
Ti3C2TX- 1.801 0.01 0.01 5.037 3.524 When the surface is coated with a silane coupling agent, MLF-NH2 ex­
MXene
NiFe-LDH 25.333 0.121 0.121 12.758 10.709
hibits the same weight loss trend as MLF at below 350 ◦ C, but an addi­
ML 123.345 0.419 0.439 23.057 13.797 tional mass loss of about 3.0 wt% occurs between 350 and 600 ◦ C, which
MLF 114.252 0.362 0.369 15.391 11.209 is possibly a result of the thermal decomposition of APTES. Therefore,
VBJH——Adsorption and desorption cumulative volume of pores； we can conclude that the silane coupling agent was successfully grafted
DBJH——Adsorption and desorption average pore size. on the MLF surface. The weight loss curve of MLF-Lac showed an
additional mass difference of about 2.1% compared to MLF-NH2, which
was attributed to the immobilization of the laccase on the support sur­
face and the decomposition of the crosslinker glutaraldehyde. Data

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M. Li et al. Colloids and Surfaces A: Physicochemical and Engineering Aspects 660 (2023) 130820

Fig. 4. (a) The XRD pattern of the NiFe-LDH, ML and MLF; (b) The FTIR spectrum of MLF and MLF-NH2.

Fig. 5. The comparison of the XPS survey scan of MLF and MLF-NH2 (a) and the high resolution XPS spectrum of (b) Ti 2p, (c) Ni 2p, (d) Fe 2p, (e) Si 2p and (f) N 1 s
of MLF-NH2.

analysis of TGA confirmed the successful preparation of immobilized 23.1 emu/g. After functionalization of the MLF surface with amino
laccase MLF-Lac. The surface potentials of the materials were further groups, the MLF-NH2 has a saturation magnetization of 16.3 emu/g.
studied (Fig. S4). After APTES modification, the negative charge carried Such damped magnetization is likely a result of the coated silane
by the surface of MLF-NH2 is enhanced, which further indicates the coupling agent. After immobilizing the enzyme, we noted that a multi­
successful modification of APTES. Laccase is negatively charged when layer coating was formed at the surface of MLF, which further reduced
the substrate pH is greater than the isoelectric point, so there is an the magnetic properties of the material.As can be seen from the inset,
electrostatic attraction between MLF-NH2 and laccase. Moreover, the although the saturation magnetization of the immobilized enzyme is
immobilization of labeled enzymes on the surface of nanocomposites 9.5 emu/g lower than that of MLF, the magnet could easily separate the
was visualized under CLSM. The observations were documented by la­ nanocomposites from aqueous solution under the intervention of an
beling laccase. A clear green fluorescence can be observed in the image external magnetic field, shows a compelling application prospect.
of MLF-Lac in Fig. S3, indicating that the laccase labeled by FITC stain is
uniformly immobilized on the surface of the vector.
The magnetization of MLF, MLF-NH2 and MLF-Lac were investigated 3.2. Optimization of laccase immobilization
by a vibrating sample magnetometer (Fig. 6b). It can be observed that
MLF nanoparticles had a relative strong magnetism, approximately Glutaraldehyde plays an important role in the immobilization of the
enzyme as a bridge between the enzyme molecule and the carrier.

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M. Li et al. Colloids and Surfaces A: Physicochemical and Engineering Aspects 660 (2023) 130820

Fig. 6. (a) TGA curves of MLF, MLF-NH2 and MLF-Lac; (b) Magnetic hysteresis loop of MLF, MLF-NH2 and MLF-Lac.

Fig. 7. Optimization of immobilized enzyme conditions: (a) glutaraldehyde concentration; (b) mass ratio of enzyme to carrier; (c) immobilization time.

Fig. 7a shows the relationship between glutaraldehyde concentration increase the amount of glutaraldehyde, the excessive cross-linking agent
and enzyme loading and relative activity of immobilized enzyme. When produced a large intramolecular or intermolecular cross-linking with the
the GA concentration is 0.0% (i.e., the immobilization method is amino groups of the enzyme, resulting in a decrease in the binding
adsorption), the relative activity of MLF-Lac is only 30.5%. With the ability of the enzyme and carrier, while the tightly cross-linked enzyme
increase of the mass fraction of glutaraldehyde, the enzyme loading and aggregates might mask or change the original structure of the enzyme,
relative activity of the immobilized enzyme increased first and then leading to a decrease in relative enzyme activity. The results showed
decreased. This may be due to the fact that as the glutaraldehyde mass that the optimum concentration of glutaraldehyde was 2.0%.
fraction increases, more enzyme molecules are covalently bound to the The initial laccase concentration can influence the relative enzyme
surface of MLF-NH2 through the Schiff’s base reaction to form amide activity of MLF-Lac nanocomposites. As the content of laccase increases,
bonds. When the concentration of glutaraldehyde reaches 2.0%, the as shown in Fig. 7a, the loading of laccase gradually increases. Conse­
relative activity of the immobilized enzyme reached its maximum value, quently, the relative activity of the MLF-lac reaches the maximum when
at which time the enzyme loading was 144.1 mg/g. Continuing to 300 mg of laccase was introduced. At this point, the enzyme load

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M. Li et al. Colloids and Surfaces A: Physicochemical and Engineering Aspects 660 (2023) 130820

reached 157.1 mg/g. However, the relative activity began to degrade laccase activity and shows that the optimal temperature for both free
with a further increase of laccase loading. The reason could be that the and immobilized laccase are both 30 ◦ C. Immobilized laccase sustained
overcrowded laccase destabilizes its conformation, and thus blocks or 60.02% of the peak activity when the temperature reached 60 ◦ C,
deactivates some of the active centers. The relative activity of the meanwhile the free laccase shows only 19.04% of the activity at the
immobilized laccase is enhanced with the immobilization time (Fig. 7b). same temperature. Enzymes are easily denatured at high temperatures,
However, the activity of the immobilized enzyme reached the maximum which indicates that the secondary and tertiary structures of free
at 3 h of the immobilization time and began to drop thereafter. This enzyme molecules undergo irreversible changes at high temperatures.
trend could be a result of the long-term shaking that can cause dena­ However, the temperature sensitivity of immobilized laccase is signifi­
turation of laccase in solution, or the overcrowded accumulation of cantly lower than that of free laccase due to the beneficial effects of the
laccase changes its stable conformation[40]. carrier that stabilizes the conformation of the enzyme and its structure at
We can conclude that the optimal duration for laccase immobiliza­ high temperatures.
tion is 3 h with the loading efficiency of 167.9 mg/g. The immobiliza­
tion efficiency and the relative enzyme activity as achieved in this work 3.3.2. Stability of free and immobilized laccase
are superior to the reported data in the prior literature. As shown in Denaturants can cause protein denaturation and therefore affect the
Table 2, compared with the immobilized carriers reported in the liter­ practical application of laccase. Different concentrations of urea were
atures, MLF-NH2 exhibited better performance. For the design of mag­ prepared as denaturants to measure the organic solvent tolerance of free
netic carriers, MLF-NH2 in this work used a two-dimensional material as laccase and immobilized laccase. Fig. 8c shows the relationship between
the main framework, which provided a broader space for the immobi­ the relative activities of free laccase and immobilized laccase as a
lization of enzyme molecules and substrate diffusion by coupling mag­ function of denaturant concentration. When the denaturant concentra­
netic particles with two-dimensional nanomaterials. While other tion increases, the relative activity of the enzyme is significantly
magnetic carriers regarded magnetic nanoparticles Fe3O4 as the main reduced. Generally, however, the degree of degradation of the immo­
body for structural modification and surface modification, however, the bilized laccase is much lower than that of the free laccase. When the urea
particle size of Fe3O4 restricted the active surface of these carriers. concentration is 1 mol⋅L− 1, the relative activity of is 81%, for the
In the present work, the higher enzyme loading of MLF-NH2 was immobilized laccase, but is only 57% for the free laccase. Continuing to
mainly attributed to two reasons. Firstly, the porous mesh structure and increase the urea concentration to 7 mol⋅L− 1, we noticed that the rela­
high specific surface area formed by the coupling of Ti3C2TX-MXene tive activity of the immobilized laccase still retains 20%, In contrast, free
nanosheets with NiFe-LDH provided a wide space for laccase adsorption laccase, as common protein, completely lost its activity. Urea denatures
and maintenance of the three-dimensional conformation, while the proteins through destructing hydrophobic interaction by increasing the
larger reaction area increased the mass transfer area and reduced the solubility of hydrophobic amino acids and therefore destroys the
adverse effect of substrate diffusion limitation on the enzyme molecules. structure of protein[45].
Secondly, the coating of APTES provided a large amount of active amino Stability is a critical part of the research on the enzymatic properties
groups, which provided a bioaffinity microenvironment for the immo­ of immobilized enzymes. Free and immobilized laccase were cultivated
bilization and enzymatic reaction of laccase. at 60 ◦ C for 7 h to assess their thermal stability and the results were
displayed in Fig. 8d. Owing to the destruction of the secondary and
3.3. Enzymatic activity of free laccase and immobilized laccase on MLF- tertiary structures of the enzyme by the high temperatures, free laccase
Laccase almost lost all its activity compared with its initial activity after 7 h. In
contrast, the thermal stability of immobilized laccase was improved and
3.3.1. Effect of pH and temperature on free and immobilized laccase retained 20% of activity after 7 h. It could be interpreted as the result of
The pH and temperature serve as important roles in determining the the combined action of various forces, such as hydrogen bonds, elec­
enzyme activity. We found from Fig. 8a that immobilized laccase shows trostatic force, and coordination bonds, which inhibited the stretch
a much higher stability in a wider pH condition than the free laccase at deformation of laccase molecules to maintain laccase activity[40].
room temperature. The free enzyme is at an optimal pH of 4.0, while the
immobilized enzyme is at ~5, MLF-laccase shows an improvement of 3.4. Michaelis-Menten kinetics of free and immobilized laccase
activity at pH > 4. This phenomenon can be well explained by the theory
that the immobilized enzymes have a more stable conformation due to The Michaelis constant Km is the basic constant of enzyme kinetics
its multipoint attachment with their supporting materials, which have [46]. It represents the concentration of the substrate when the enzyme
been well reported elsewhere[44]. The laccase is connected to the MLF reaction rate reaches half of the maximum reaction rate and reflect the
through the bifunctional cross-linking reagent glutaraldehyde. Such affinity of the enzyme to the substrate[33]. The Km and the maximum
feature reduces the molecular mobility of the enyzme. Additionally, the velocity of enzyme-catalyzed reaction (Vmax) of free laccase and
large carrier surface as obtained by MLF provides multiple attachment immobilized laccase were calculated using the Line weaver-Burk plots
sites for laccase to maintain its conformation, enabling the enzyme to (Fig. 9). Free laccase presents an apparent Km of 0.0758 mM and a
resist acid and alkaline adverse environments. maximum reaction rate Vmax of 0.4889 mM/min. The immobilized
Fig. 8b compares the effect of temperatures on free and immobilized enzyme MLF-lac presents an apparent Km of 0.1401 mM and a maximum
reaction rate Vmax of 0.2122 mM/min. The Michaelis constant Km of the
immobilized enzyme is obviously higher than that of the free enzyme,
Table 2
indicating that the affinity of the immobilized enzyme to the substrate is
Comparison of loading capacity and enzyme activity recovery rate of various
composite materials immobilized enzymes. smaller than free enzyme. This could be related to the steric hindrance of
the immobilized carrier to the catalytic activity center of the enzyme. In
Supporting Bound enzyme (mg/ Activity retention References
addition, the two-way diffusion of the substrate and the reaction product
g) (%)
is also restricted by steric hindrance, resulting in a decrease in the cat­
Fe3O4-PDA 171 51.00 [32]
alytic ability of the immobilized laccase[47].
Fe3O4 @MoS2 @PEI 120.00 90.00 [33]
PDA/HNTs 41.28 82.67 [41]
Fe3O4-NH2-Cu2+ 76.03 42.74 [42] 3.5. Degradation of organic phenol by free and immobilized laccases
Fe3O4 @SiO2- 158 79.10 [43]
chitosan In this study, catechol, chlorophenols 2,4-dichlorophenol (2,4-DCP)
MLF-NH2 167.91 76.92 This work
and polycyclic aromatic hydrocarbons bisphenol A (BPA), which are

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M. Li et al. Colloids and Surfaces A: Physicochemical and Engineering Aspects 660 (2023) 130820

Fig. 8. Effect of (a) pH (b) temperature on the activity of the free and MLF-lac; (c) Effect of urea with different concentrations and (d) the thermal stability (at 60 ◦ C)
on the stability of the free and immobilized laccase.

of 93.94%, 95.76% and 100% for the three organic phenols,

respectively.
The degradation rates of free enzymes and immobilized enzymes to
three organic phenols in different reaction durations are shown in the
Fig. 10b, c. High degradation rate were observed in both the initial 4 h.
However, the removal efficiency of organic phenols by free and immo­
bilized laccase gradually decreased in the following response period,
which might be attributed to the decrease in the concentration of three
organic phenols that available for the reactions.
Excellent reusability is essential for practical applications. To reduce
the material cost, the magnetic immobilized enzyme can be separated by
a magnet after the reaction and reused. From Fig. 10d-f, after seven
repeated runs, approximately 55.49%, 92.11% 70.98% of removal ef­
ficiency could be retained for immobilized laccase according to 2,4-DCP,
BPA and pyrocatechol, respectively. In addition to excellent catalytic
performance, the catalytic material also needs to show excellent
adsorption performance to pre-enrich organic matter locally near the
surface of the catalyst for efficient phenol degradation. Therefore, we
Fig. 9. The Lineweaver-Burk plots of free and immobilized laccase;. compared the changes in the adsorption capacity of the carrier for
organic phenols during the repeated utilization process. MLF-NH2
common organic phenols in industrial wastewater, were selected as nanocomposite could also remove 21.41% 2,4-DCP, 44.98% BPA,
target substrates to evaluate the enzymatic activity. Laccase is usually 14.58% pyrocatechol only by surface adsorption. The decreased degra­
used to study the removal of organic phenol in water on account of its dation efficiency of immobilized enzyme in the process of reusability
unique advantages, especially avoiding secondary pollution[48]. In likely due to (1) the congestion of reaction products which block the
principle, the laccase is able to remove organic phenol by oxidizing it to channels and hinder the access to substrates[32] and/or (2) the enzyme
form active radicals, which can polymerize the phenols to polymers that detachment and deactivation during constant mechanical stirring[49].
can be precipitated with low toxicity from water. Fig. 10a compared the
removal efficiency of 2,4-DCP, BPA, and pyrocatechol in water by car­ 3.6. Comparison of the performance of some immobilized laccase
rier MLF-NH2, free laccase and MLF-lac at 25 ◦ C. The removal efficiency
of free laccase for 2,4-DCP, BPA, and pyrocatechol is 75.34%, 90.66% The comparison results of different organic compounds removal
and 97.73%, respectively, while MLF-lac exhibits the removal efficiency performance of laccase immobilized on various carriers are summarized

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M. Li et al. Colloids and Surfaces A: Physicochemical and Engineering Aspects 660 (2023) 130820

Fig. 10. (a) Removal efficiency of 2,4-DCP, BPA and pyrocatechol catalyzed by carrier, free laccase and immobilized laccase; Removal efficiency of 2,4-DCP, BPA
and pyrocatechol catalyzed by (b) free laccase and (c) immobilized laccase in different time periods; Reusability of immobilized laccase and MLF nanocomposite in
the removal of (d) 2,4-DCP, (e) BPA, (f) pyrocatechol. Experimental conditions: 2,4-DCP (10 mg/L), BPA (10 mg/L), Pyrocatechol (10 mg/L), pH7.0, 30 ℃,
10 mL, 12 h.

Table 3
Comparison of organic compounds removal performance of laccase immobilized on various carriers.
Laccase source Immobilized Substrate Degradation (%) References
enzyme

Trametes PP-co-pGMA-laccase Crystal Violet (CV), Procion Green H 4 G (PG-H4G), Brilliant Blue G 99%, 100%, 90% [50]
versicolor (BBG)
Trametes PS-co-DVB-g-P(CCMA)-laccase Bisphenol A, 89%, 100% [51]
versicolor Congo Red dye
Trametes p(MMA-EGMA-CCMA)-laccase Methylene Blue dye, Carbaryl pesticide 63%, 71% [52]
versicolor
Trametes p(HEMA-co-VC)-laccase Cibacron Blue 3GA (CB3GA) 100% [53]
versicolor
Trametes PET/ABTS@UiO-66(Zr)-NH2- Crystal Violet (CV), 58.8%, 64.8%, 61.6%, [54]
versicolor laccase Malachite Green (MG), 21.1%
Alizarin Green (AG),
Methyl Orange (MO)
Trametes laccase-AS@BC/GMA-DA 2,4,5-trichlorophenol 98.3% [55]
versicolor

in Table 3. It is obvious from Table 3 that immobilized laccase has a very surfaces by cross-linking agents with a loading of 167.9 mg/g. The MLF-
wide range of applications in the degradation of phenolic pollutants, Lac showed better tolerance to acidic/alkaline acids, high temperature
polycyclic aromatic hydrocarbons and dyes. The ability of laccase to and organic solvents compared with free laccase, and excellent opera­
catalyze a variety of aromatic compounds makes it attractive for in­ tion stability. Moreover, the degradation efficiency of MLF-Lac for 2,4-
dustrial applications. DCP, BPA, and pyrocatechol was 93.94%, 95.76% and 100% within
12 h, which was higher than that of free laccase. We expect that the
4. Conclusions nanoreactor with superparamagnetic properties can provide more ideas
for the application of high-performance biocatalysts.
To sum up, the novel MXene@NiFe-LDH@Fe3O4 nanocomposites
were successfully prepared through the heterogeneous assembly of two- CRediT authorship contribution statement
dimensional Ti3C2TX-MXene nanosheets, layered double hydroxides
(LDHs) and magnetic nanoparticles. Afterwards, covalently connected Mengyu Li： ： Writing – original draft, Validation, Formal analysis,
fixed laccase through interface modification. The results showed that the Visualization, Methodology. Yahan Bai: Data curation, Investigation.
MXene@NiFe-LDH@Fe3O4 nanocomposites exhibited a larger specific Wei Zhuang： ： Methodology, Writing – review & editing, Supervision,
surface area of 114.3 m2/g, providing more attachment sites for the Resources, Funding acquisition. Jinle Liu: Project administration,
enzyme molecules. The coating of APTES provides reactive groups for Methodology. Zhi Wang: Resources, Data curation. Yuan Rao: Formal
nanocomposites, and laccase was successfully immobilized on their analysis, Data curation, Validation. Mengran Li: Investigation,

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M. Li et al. Colloids and Surfaces A: Physicochemical and Engineering Aspects 660 (2023) 130820

Visualization. Hanjie Ying: Funding acquisition, Resources. Pingkai [18] J. Zdarta, A.S. Meyer, T. Jesionowski, et al., Developments in support materials for
immobilization of oxidoreductases: a comprehensive review, Adv. Colloid Interface
Ouyang: Funding acquisition.
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Catalysts 8 (2) (2018) 92.
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interests or personal relationships that could have appeared to influence nitrides (MXenes): characteristics, environmental remediation and challenges,
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performance polymer composites: a review, Compos. Part B: Eng. 217 (2021),
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