Construction of a bioinspired laccase-mimicking nanozyme for the degradation and detection of phenolic pollutants
Graphical abstract
Introduction
Phenolic compounds such as chlorophenols and bisphenols are common toxic pollutants due to their broad use in wood preservatives, pesticides and disinfectants [1]. These phenolic compounds, regarded as threats to human health via carcinogenesis, reproductive toxicity, neurotoxicity and endocrine disruption, are widely detected in ground soil and water [2]. Laccases are members of the multi-copper oxidases (MCOs), which catalyze the single-electron oxidation of a wide range of organic substrates, such as polyamines, aryl diamines, ortho- and para-diphenols as well as polyphenols, with the subsequent four-electron reduction of molecular oxygen to water [[3], [4], [5], [6]]. Therefore, laccases can be utilized as green catalysts in water treatment and soil bioremediation [7,8]. Nevertheless, the poor stability of the laccases in complex environments and the difficulty in recycling laccases severely hamper its practical application due to the high cost. To achieve the recyclability of laccases, many efforts have focused on the immobilization of the laccases on suitable matrices [[9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20]]. For example, Sarma et al. assembled laccase layer-by-layer on stimuli-responsive membranes for chloro-organic degradation which displayed a loss of 14% of its initial activity after four cycles of operation [14]. Ji et al. immobilized a laccase on a mediator membrane hybrid reactor for the biocatalytic degradation of carbamazepine which maintained 15% of its initial activity after 5 degradation cycles [12]. Recently, Li et al. immobilized a laccase on a Cu2O nanowire mesocrystal material for the bioremediation of 2,4-dichlorophenol-contaminated water with high biocatalytic activity, and the efficiency remained at 75% after 10 cycles [15]. Although the problem of recyclability was solved by enzyme immobilization, the stability of the enzymes is still one of the most difficult problems in protein chemistry due to the intrinsic fragility of the enzyme under complex conditions.
To obtain a highly stable biocatalyst, the construction of a nanozyme that mimics the natural enzyme is a promising strategy [21]. Nanozymes, defined as nanomaterials with enzyme-mimicking activity [22], have attracted extensive research interest due to their multifunctionality, low cost and high stability [23]. A variety of nanomaterials and their derivatives, such as noble metals [[24], [25], [26], [27], [28], [29], [30], [31], [32]], metal oxides [[32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42]] and carbon-based nanomaterials [[43], [44], [45]], were developed as nanozymes in the fields of virus detection [29], the detection of heavy metal ions [46], degradation of organophosphorus-based nerve agents [33] and bacterial inactivation [32,47]. Compared with natural enzymes, nanozymes show better stability and durability due to the inherent properties of the nanomaterial [48,49]. For example, Kuo et al. prepared Au@Cu2O core@shell nanocrystals as dual-functional catalysts for sustainable environmental applications and the efficiency of methyl orange degradation remained at 89% after 4 cycles [32]. Wang et al. prepared vanadate quantum dots-interspersed g-C3N4 nanocomposites for efficient inactivation of Salmonella by harvesting visible light [47]. More recently, organic-inorganic hybrids based on the coordination of metal-based nodes with organic ligands have appeared as a promising class of nanozymes owing to their facile preparation, appropriate size and biocompatibility. For example, Liang et al. prepared a nanozyme with laccase-like activity based on guanosine monophosphate (GMP)- coordinated copper [50]. Wang et al. synthesized a porphyrinic metal organic framework PCN-600 (Fe) which is stable under aqueous conditions and has the catalytic activity of peroxidase [51]. Furthermore, this method has been indicated to be effective in mimicking the coordination microenvironment of enzymatic active sites for the design and synthesis of nanozymes with high catalytic activity. Because the structure of the active site and reaction mechanism of peroxidases is well studied [52], a large body of work has focused on the preparation of nanozymes with the catalytic activity of peroxidase [37,[51], [52], [53]]. For instance, Fan et al. optimized the peroxidase-like activity of the Fe3O4 nanozyme via a single amino acid modification to mimic an enzyme active site [37].
Compared with the research of nanozymes with peroxidase-like activity, there are limited reports on nanozymes with laccase-like activity [50,54,55], which is probably due to the complex structure of the active site and catalytic mechanism of laccase. Specifically, there are four copper sites in laccases that include Type 1 (T1), Type 2 (T2) and binuclear Type 3 (T3) Cu sites. The oxidation of the substrates occurs at the T1 Cu site, and electrons transfer from the T1 Cu site to the T2/T3 trinuclear Cu cluster where the molecular oxygen converts to water through a cysteine-histidine (Cys-His) pathway [5]. Inspired by the structure of the active site and the electron transfer pathway of laccase, we attempted to design and synthesize nanomaterials utilizing a Cys-His dipeptide coordinated with copper ions to mimic this catalytic process.
Herein, we present a facile strategy for the preparation of a new class of nanozyme (denoted as CH-Cu) with laccase-like activity. This strategy is inspired by the structure of the active site and the electron transfer pathway of laccase via coordination of Cu+/Cu2+ with a Cys-His dipeptide. Specifically, the CH-Cu nanozymes were synthesized through a hydrothermal method using Cys-His dipeptide and CuCl2 as precursors. The structural characterization of the CH-Cu nanozymes was performed using SEM, FTIR, XPS and XRD. The catalytic activity, stability, recyclability and substrate specificity of the CH-Cu nanozymes were evaluated and compared to the natural enzyme. Additionally, the quantitative detection of epinephrine by a smart phone was established based on the CH-Cu nanozymes. Finally, the catalytic mechanism of action for the CH-Cu nanozymes was proposed.
Section snippets
Materials
The cysteine-histidine dipeptide, cysteine and histidine were purchased from GL Biochem (Shanghai) Ltd. 2,4-dichlorophenol (2,4-DP), 4-aminoantipyrine (4-AP), 2,2′-azino- bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2-(N-morpholino)ethanesulfonic acid (MES) monohydrate, N,N-dimethylformamide (DMF) and copper (II) chloride dihydrate were obtained from Aladdin Industrial Corp. (Shanghai, China). Laccase from Trametes versicolor and horseradish peroxidase (HRP) were purchased from
Results and discussion
Scheme 1 illustrates the synthesis of the natural laccase-inspired CH-Cu nanozymes. The Cu2+ and Cys-His dipeptide, which are the key intermediates of the electron transfer in the natural laccase combination of the substrate oxidation with oxygen reduction [4,6], were used to prepare the nanozyme by a hydrothermal method. The Cys-His dipeptide could be utilized as metal ligands to form a metal-organic framework (MOF) analogy with metal ions because both the thiol group in cysteine and the
Conclusions
In summary, we have successfully prepared a new CH-Cu nanozyme inspired by the structure of the laccase active site. Compared with laccase, the CH-Cu nanozymes shows higher catalytic activity under normal conditions and greater stability at extreme pH, temperature, after long-term storage and at high salt concentration. The CH-Cu nanozymes also display good recyclability. Acceptable substrate universality was observed in the CH-Cu nanozymes, enabling us to utilize it for chlorophenol and
Acknowledgments
This work was supported by the National Natural Science Foundation of China (Nos. 21621004, 51773149 and 21777112).
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