Construction of a bioinspired laccase-mimicking nanozyme for the degradation and detection of phenolic pollutants

Highlights

• An active site-inspired CH-Cu nanozyme with laccase-like activity is synthesized.

• The CH-Cu nanozyme has superior activity and stability compared to natural enzyme.

• The CH-Cu nanozyme has broad substrate specificity for phenolic pollutants.

• The CH-Cu nanozyme maintains 82% of its catalytic activity after twelve cycles.

• The detection of epinephrine by a smart phone is established based on the CH-Cu nanozyme.

Abstract

Nanozymes, defined as nanomaterials with enzyme-like activity, have attracted extensive interest in both fundamental and applied research. Laccases are members of the multi-copper oxidases, which are utilized as green catalysts in the environmental catalysis and biochemical industry. In this paper, we report a facile strategy for the preparation of a new class of nanozyme (denoted as CH-Cu) with laccase-like activity inspired by the structure of the active site and the electron transfer pathway of laccase via the coordination of Cu⁺/Cu²⁺ with a cysteine (Cys)-histidine (His) dipeptide. The CH-Cu nanozymes exhibit excellent catalytic activity, recyclability and substrate universality and have a similar K_m (Michaelis constant) and a higher v_max (maximum rate) than laccase at the same mass concentration. They are robust under a variety of conditions, such as extreme pH, high temperature, long-term storage and high salinity, which can cause severe loss in the catalytic activity of laccase. Higher efficacy of the CH-Cu nanozymes compared with laccase in the degradation of chlorophenols and bisphenols is also demonstrated in a batch reaction. Furthermore, a method for the quantitative detection of epinephrine by a smart phone is established based on the CH-Cu nanozymes. We believe that this nanozyme has promising applications in environmental catalysis and rapid detection and expect that combining key peptides as metal ligands with metal ions to mimic the structure of the catalytic center of a natural enzyme will be a general and important strategy for the design and synthesis of a new type of nanozyme that can be used in various applications.

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.