Construction of novel bienzyme-inorganic hybrid nanoflowers beads and their application in the efficient degradation of acridine

Yaohua Gu; Lin Yuan; Mingming Li; Ying Liu; Xiaoyan Bai; Keren Shi; Mengling chen; Huiqin Yao

doi:10.1016/j.arabjc.2023.104770

View/Download PDF

Buy Reprints

PDF

Translate this page into:

Original article

06 2023

:16;

104770

doi:

10.1016/j.arabjc.2023.104770

Construction of novel bienzyme-inorganic hybrid nanoflowers beads and their application in the efficient degradation of acridine

Yaohua Gu^{^a,1}, Lin Yuan^{^a,1}, Mingming Li^{^b,1}, Ying Liu^{^a}, Xiaoyan Bai^{^a}, Keren Shi^{^c}, Mengling chen^{^a}, Huiqin Yao^{^a,⁎}

a Key Laboratory of Environmental Factors and Chronic Disease Control, College of Public Health and Management, Ningxia Medical University, Yinchuan 750004, PR China

b Urology Surgery, General Hospital of Ningxia Medical University, Yinchuan 750004, PR China

c State Key Laboratory of High-efficiency Coal Utilization and Green Chemical Engineering, Ningxia University, Yinchuan 750021, PR China

⁎Corresponding author. yaohq@nxmu.edu.cn (Huiqin Yao)

Received: 2022-12-30, Accepted: 2023-2-27,

Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

1 Contribute equally as the first author.

Abstract

To develop a biocatalyst with high efficiency, stability and reusability for the degradation of organic pollutant acridine in wastewater, a novel biocatalyst named bienzyme-inorganic hybrid nanoflowers beads were successfully prepared in the present work, designated as biE-HNFs-beads. This bienzyme biocatalyst composed of bienzyme-inorganic hybrid nanoflowers and traditional beads carriers were characterized by SEM, TEM, FT-IR, XRD, and EPR. The biE-HNFs-beads showed better stability, recyclability and improved catalytic activity for acridine degradation than that of free bienzyme. Furthermore, biE-HNFs-beads could reduce the diffusion and decomposition of the intermediate H₂O₂ through the bienzyme cascade reactions, which not only improved the stability of the bienzyme, but also accelerated the degradation of acridine. Under optimized conditions, the degradation rate of acridine by biE-HNFs-beads could reach 99.9 %. The stability study indicated that the biE-HNFs-beads exhibited enhanced stability, reusability and storage stability. Besides, the bioconversion products analysis of acridine degraded by biE-HNFs-beads biocatalyst were identified by LC-MS, and the enzymatic degradation pathway of acridine was inferred for the first time.

Keywords

Biocatalysis

Cascade reaction

Acridine degradation

Wastewater treatment

Bienzyme-inorganic hybrid nanoflowers beads;

Show Related Articles from PubMed

1 Introduction

With the rapid development of economy and the continuous increase of the chemical industry, the content of refractory organics in wastewater gradually increases, which poses a severe threat to the ecological environment and human health (Kaiser et al., 1996). Acridine is a typical nitrogen heterocyclic compound, which is widely used in cooking, pharmaceutical, dye and other processes (Abdelhameed et al., 2019), and it has carcinogenicity, teratogenicity and mutagenicity. Most importantly, its chemical structure is stable and difficult to eliminate under natural conditions. Therefore, how to remove acridine from wastewater is a research hotspot at present.

So far, many methods have been applied to the treatment of industrial wastewater, such as physical method, chemical method, microbial method and bio-enzyme method (Wang et al., 2015; Arif et al., 2018; Li et al., 2021; Fang et al., 2022; Mushtaq et al., 2022 ). However, methods for removing acridine from wastewater are limited and inefficient. Inspired by the fact that enzyme-redox medium system can improve enzyme catalytic activity, we consider using biological enzyme-redox medium system to catalyse the degradation of acridine in wastewater. Enzyme catalysis is a kind of green, safe, and high-efficiency biodegradation technology. Up to now, horseradish peroxidase (HRP), laccase and other enzymes have been used to degrade organic pollutants in wastewater (Razzaghi et al., 2022). Among them, HRP is considered an effective strategy to degrade pollutants for its high activity, non-toxicity and mild reaction conditions (Ocsoy et al., 2015; Altinkaynak et al., 2021). HRP is a classical heme enzyme, which can catalyse organic contaminants to form small molecule structures in the presence of hydrogen peroxide (H₂O₂) (Li et al., 2018b). Nevertheless, H₂O₂ is corrosive and irritating, imposing an additional burden on the environment (Hiner et al., 2001). Recently, inspired by the bienzyme cascade reaction system (Sharma et al., 2017), we combined glucose oxidase (GOD) with HRP into a bienzyme system, where GOD catalyses glucose (Glu) to produce H₂O₂ for subsequent HRP biocatalysis (Huang et al., 2015). In contrast to the single enzyme system, the bienzyme system performs the cascade reaction in a stepwise fashion without separating the intermediates. The advantage of this method is that, on the one hand, it can reduce the harm caused by the intermediate H₂O₂ to the environment and the bienzyme, and improve the bienzyme stability based on green and safe. On the other hand, it is beneficial to improve the efficiency of the reaction by reducing the diffusion of intermediate H₂O₂ (Zhu et al., 2017).

The application of enzyme is limited by their high price, low stability and high solubility (Zhang et al., 2022). Enzyme immobilization is an effective strategy to solve these problems, which improves enzyme activity, stability and reusability (Zhu et al., 2013). The structure and properties of the immobilized carriers also affect the performance of the enzyme to some extent (Cui et al., 2018b; Fu et al., 2019; Du et al., 2022). With the continuous development of nanotechnology, nanomaterials have been widely used as carriers for immobilized enzyme (Li et al., 2018a). In 2012, Zare’s group (Ge et al., 2012) initially discovered nanoflowers biocatalyst by co-precipitating Cu₃(PO₄)₂ and enzyme in a PBS buffer solution. These nanoflowers presented a complete flower-like structure in SEM images. Different from traditional immobilization methods, the nanoflowers biocatalyst synthesized by biomineralization has the advantages of large surface area, high activity, simple operation, green safety and so on (Cui and Jia, 2017; Li et al., 2020; Zhong et al., 2021 ). However, nanoflowers are powdery and have poor mechanical strength, which can easily lead to the destruction of flower-like structures and poor reusability during the separation process (Ren et al., 2019). These shortcomings hinder their application in industry. Therefore, it is an essential attempt to increase the enzymatic properties of nanoflowers biocatalyst with high mechanical strength and good stability carrier. (Lee et al., 2017; Zhao et al., 2017; Cui et al., 2016; Wen et al., 2020 ).

Cellulose-chitosan (Ce-Cs) beads have good biocompatibility, functional and mechanical properties, and are widely used as carrier materials for enzyme immobilization. Dopamine (DA) from mussels can self-polymerize to form polydopamine (PDA) in alkaline environment (Mohammad et al., 2020). The universal and firm adhesion of PDA provides a powerful platform for modifying a wide range of carrier materials (Jiang et al., 2018). In aqueous solution, PDA can concentrate and adsorb metal ions on the surface of Ce-Cs beads, and combine with enzyme molecules to form the growth site of nanoflowers through biomineralization, so that nanoflowers grow firmly on the surface of the beads. Therefore, the Ce-Cs beads modified by DA are good nanoflowers carrier. To our knowledge, there have been no similar studies on such nanoflowers beads.

In this study, we attempted to combine the bienzyme-inorganic hybrid nanoflowers (biE-HNFs) with the Ce-Cs beads to prepare a supported nanoflowers biocatalyst, designated as bienzyme-inorganic hybrid nanoflowers beads (biE-HNFs-beads). To verify the successful synthesis of biE-HNFs-beads, the methods of SEM, TEM, EDS, XRD, FT-IR and EPR were used to characterize biE-HNFs-beads. Besides, to explore the optimal synthesis conditions, incubation temperature, bienzyme ratio and different preparation methods for forming biE-HNFs-beads were discussed. To explore the best reaction conditions, the effects of substrate concentration, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) dosage, Glu dosage on the degradation rate of acridine were investigated. Furthermore, the stability, reusability and storage stability of the biE-HNFs-beads were systematically evaluated. The results proved that the stability, reusability and storage stability of the biE-HNFs-beads were better than those of free bienzyme. This study provides an essential innovation for the preparation of active, stable and recyclable carrier-supported nanoflowers biocatalyst, which can be used for the efficient degradation of acridine in wastewater.

2 Materials and methods

2.1

2.1 Chemicals and enzyme

Horseradish peroxidase (HRP, greater than200 U/mg) was purchased from TCI Development Co., Ltd. (Shanghai, China). Glucose oxidase (GOD, greater than100 U/mg), acridine (purity 98.0 %) and glucose (Glu) were provided by Sigma (St. Louis, MO, USA). Microcrystalline cellulose (Ce), chitosan (Cs), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 1-ethyl-3-methylimidazolium acetate ([Emim] [OAc]) were all purchased from Hengyuanchuang Technology and Trade Co., Ltd. (Ningxia, China). Copper sulfate pentahydrate (CuSO₄•5H₂O), Tris(hydroxymethyl)aminomethane (Tris) and dopamine hydrochloride (DA) were all obtained from Innochem Technology Co., Ltd. (Beijing, China). HPLC-grade methanol was supplied by Fisher Company (Morristown, NJ, USA). All the chemicals used were of analytical grade purity.

2.2

2.2 Preparation of biE-HNFs-beads

2.2.1

2.2.1 Preparation of Ce-Cs@DA beads

The Ce-Cs beads were prepared according to our previous work (Gu et al., 2022). In a typical synthesis, Ce (0.3 g), Cs (0.3 g) and [Emim] [OAc] (10 mL) were placed in a three-neck round-bottom flask (25 mL) and stirred at 1000 rpm at 85 °C for 3 h. Ce-Cs beads were obtained by dropping the synthesized mixture into ultrapure water using a 10 mL plastic syringe with a 26-gauge needle. After hardening treatment for 2 h, the beads were washed three times in ultrapure water.

Subsequently, Ce-Cs beads (1.0 g) and DA (15 mg) were charged into a Tris-HCl buffer solution of pH 8.5 (10 mL, 0.01 mol/L) and stirred at 150 rpm at 30 °C for 3 h to obtain Ce-Cs@DA beads. Then, the beads were washed three times in ultrapure water.

2.2.2

2.2.2 Adsorption of copper ions on the surface of Ce-Cs@DA beads.

In order to adsorb copper ions (Cu²⁺) on the surface of Ce-Cs@DA beads, the beads (1.0 g) were immersed into CuSO₄ solution (10 mL, 0.1 mol/L) for 12 h at 30 °C. Then Cu²⁺ was adsorbed on the surface of the beads by the principle that polydopamine is easy to concentrate and adsorb metal ions (Li et al., 2016a). Afterward, the beads were purified with ultrapure water for three times to remove the free Cu²⁺, and named as Ce–Cs@DA/Cu²⁺ beads.

2.2.3

2.2.3 Preparation of biE-HNFs-beads

The synthesis of biE-HNFs-beads was performed by the conventional coprecipitation method (Li et al., 2016a). Ce-Cs@DA/Cu²⁺ beads (1.0 g) were added into PBS buffer solution (0.1 mol/L, pH 7.4) containing HRP-GOD in different proportions of concentration (3:1, 2:1, 1:1, 1:2 and 1:3, respectively). After incubation for 72 h at different temperatures (0 °C, 10 °C, 20 °C, 30 °C and 40 °C, respectively), the synthesis process was completed. Subsequently, biE-HNFs-beads were washed three times with a PBS buffer solution. In the present study, the conditions for preparing biE-HNFs-beads were optimized through a series of experiments including the synthesis temperature, the concentration ratios of HRP:GOD in solution and different preparation methods of biE-HNFs-beads.

In contrast, HRP-GOD biE-HNFs powder was prepared by adding CuSO₄ solution (0.1 mL, 0.12 mol/L) to a PBS buffer solution (0.1 mol/L, pH 7.4) containing HRP-GOD. A large amount of blue precipitate appeared in the solution after 72 h. The precipitate was centrifuged at high speed (10000 rpm, 5 min) and biE-HNFs powder was collected after freeze-drying.

Additionally, HRP@GOD-HNFs-beads, GOD@HRP-HNFs-beads, HRP-HNFs-beads, and GOD-HNFs-beads were prepared to study the effects of different preparation methods of biocatalyst on the formation of the beads-supported nanoflowers biocatalyst, respectively. Their preparation process was as follows. For preparing HRP@GOD-HNFs-beads and GOD@HRP-HNFs-beads, Ce-Cs@DA/Cu²⁺ beads were firstly put into a PBS buffer solution containing HRP (6.7 mg) and GOD (3.3 mg) for 36 h, respectively. Then they were removed and soaked in a PBS buffer solution containing GOD (3.3 mg) and HRP (6.7 mg) for 36 h, respectively. For preparing HRP-HNFs-beads and GOD-HNFs-beads, Ce-Cs@DA/Cu²⁺ beads were placed into a PBS buffer solution containing HRP (10 mg) or GOD (10 mg) for 72 h, respectively.

2.3

2.3 The catalytic activity and encapsulation yield of biE-HNFs-beads

According to the literature, HRP activity was determined by spectrophotometry by generating ABTS radicals (Pitzalis et al., 2017). biE-HNFs-beads and biE-HNFs were incubated with a PBS buffer solution (2.8 mL, 0.01 mol/L, pH 7.4) containing ABTS (100 μL, 0.02 mol/L) and Glu (100 μL, 0.5 mol/L), next, incubated at 30 °C for 3 min. The change in absorbance was measured with a UV–vis spectrometer at 414 nm (ε = 31100 L/(mol·cm)). The catalytic activity was determined by calculating the absorbance change in ABTS solution before and after the reaction. Among them, the maximum activity measured under the same conditions was defined as 100 %, and the ratio of the activity of tested sample to the maximum activity was its relative activity (%).

(1)

E n c a p s u l a t i o n y i e l d (%) = (C - C_{0}) / C \times 100 %

Where C and C₀ represent the concentration of bienzyme in the supernatant before and after immobilization, respectively.

2.4

2.4 Evaluation of the kinetic parameters of the biE-HNFs-beads

In order to show the effectiveness of the immobilization process, the Michaelis–Menten kinetic parameters of the biE-HNFs-beads and the free bienzyme were evaluated (Li et al., 2017). K_m and V_max can be determined by measuring the degradation rate of biE-HNFs-beads and free bienzyme to ABTS solution of different concentrations (0.3 mmol/L ∼ 20.0 mmol/L) at room temperature. K_m and V_max were calculated according to the Lineweaver–Burk double reciprocal model of the Michaelis–Menten equation as follows:

(2)

1 / V = K_{m} / V_{\max} \times 1 / [S] + 1 / V_{\max}

where V (mmol·min/L) is the apparent initial catalytic rate, V_max (mmol·min/L) is the maximum clear initial catalytic rate, [S] (mmol/L) is the substrate concentration, and K_m (mmol/L) is the apparent Michaelis–Menten constant.

2.5

2.5 Degradation of acridine by biE-HNFs-beads

The degradation efficiency of acridine by biE-HNFs-beads was determined by high-performance liquid chromatography (HPLC). The reaction solution (10 mL, pH 7.0), containing a mixture of acridine (15 mg/L), biE-HNFs-beads (1.0 g), ABTS (3.0 mg/mL) and glucose (2.0 mg/mL), was vibrated for 12 h at 30 °C. A similar procedure was adopted for the reactions by using the biE-HNFs (1.0 mg). In this case, the tests were performed for the degradation of acridine. All reactions were performed in triplicate, and data were the average of triplicate data. Besides, the degradation products of acridine were identified by liquid chromatography-mass spectrometry (LC-MS). The degradation rate of acridine can be calculated as follows:

(3)

D (%) = (C_{1} - C_{t}) / C_{1} \times 100 %

Where C₁ and C_t represent the concentration of acridine in the initial and final solution after the reaction, respectively.

2.6

2.6 The properties of biE-HNFs-beads

2.6.1

2.6.1 Thermal and pH stability of biE-HNFs-beads

In order to study the thermal stability of the biocatalyst, free bienzyme, biE-HNFs and biE-HNFs-beads were used to degrade 15 mg/L of acridine simulated wastewater at different temperatures (10 °C ∼ 60 °C) while other reaction conditions remained unchanged (pH 7.0, ABTS 3 mg/mL, Glu 2 mg/mL). After 8 h reaction, the degradation rate of each sample was determined by HPLC.

To study the pH stability of the biocatalyst, 15 mg/L acridine simulated wastewater was prepared with buffer solution of different pH (4.0 ∼ 9.0). Under other conditions unchanged (30 °C, ABTS 3 mg/mL, Glu 2 mg/mL), free bienzyme, biE-HNFs and biE-HNFs-beads were used to degrade the simulated wastewater of acridine at different pH conditions. After 8 h reaction, the degradation rate of each sample was determined by HPLC.

2.6.2

2.6.2 Storage stability of biE-HNFs-beads

To evaluate the storage stability of biE-HNFs, biE-HNFs-beads and free bienzyme, they were immersed in PBS solution at 4 °C for 2 months, respectively, and then appropriate amounts were removed every 10 days for degradation of acridine under the optimal conditions.

2.6.3

2.6.3 Reusability of biE-HNFs-beads

To investigate the reusability of biE-HNFs and biE-HNFs-beads, they were subjected to 10 consecutive acridine degradation experiments under the same operating conditions, respectively. Each cycle was carried out under optimal conditions, and the newly prepared acridine solution (10 mL, 15 mg/L) was added.

2.7

2.7 Characterization of biE-HNFs-beads

All samples were freeze-dried and characterized. The morphology of biE-HNFs-beads was studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The composition and spatial distribution of elements in biE-HNFs-beads were analyzed by energy dispersive spectroscopy (EDS). Fourier transform infrared (FT-IR) spectroscopy was used to analyze the functional groups of biE-HNFs-beads. X-ray diffraction (XRD) was studied to investigate the structure of biE-HNFs-beads. Supernatant from biE-HNFs-beads + ABTS + Glu, biE-HNFs + ABTS + Glu and free HRP-GOD + ABTS + Glu were carried out to monitor electron transfer radical signals by electron paramagnetic resonance (EPR). The specific surface area of biE-HNFs was analyzed by Brunner-Emmet-Teller (BET) measurements. The charge transfer resistance of biE-HNFs was studied by electrochemical impedance spectroscopy (EIS).

3 Results and discussion

3.1

3.1 Synthesis mechanism of biE-HNFs-beads

The traditional synthesis mechanism of biE-HNFs is shown in Fig. 1A. Briefly, the growth process of biE-HNFs consisted of three steps (Wu et al., 2016). Firstly, Cu²⁺ reacted with PO₄³⁻ in the PBS to form primary copper phosphate crystals. At the same time, the amide groups in bienzyme molecules coordinated with Cu²⁺ to form complexes, which provided a location for the nucleation of primary crystals. Secondly, the copper phosphate crystals formed a lamellar structure with a nucleation site as the growth center, that was, the primary petals. Finally, anisotropic growth led to the complete formation of branching flower structures. Herein, enzymes were crucial throughout the growth process, inducing the nucleation of copper phosphate crystals to form a scaffold for the petals and acted as the “glue” that held the petals together to form the complete nanoflowers.

Synthesis mechanism of biE-HNFs (A). Fabrication procedures of biE-HNFs-beads and their degradation mechanism of acridine (B).

The synthesis mechanism of biE-HNFs-beads with flower-like surface structure was schematically illustrated in Fig. 1B. Firstly, Ce-Cs beads were modified with DA. Secondly, Cu²⁺ was adsorbed on the surface of the beads. This is because DA was easy to self-polymerize under alkaline conditions to form polydopamine, which had substantial adhesion properties and could adsorb Cu²⁺ on the surface of beads to form stable metal-DA chelates (Zhang et al., 2011). Thirdly, PO₄³⁻ in the PBS buffer solution reacted with Cu²⁺ on the surface of the beads to form primary copper phosphate crystals. Meanwhile, the amide groups in bienzyme molecules coordinated with Cu²⁺ on the surface of the beads to form nucleation site of nanoflowers, which provided a location for the formation of the primary petals (Rong et al., 2017). Finally, through the anisotropic growth, many complete nanoflowers structures are formed on the surface of the beads.

The catalytic process of acridine degradation catalyzed by biE-HNFs-beads is presented in Fig. 1B. Firstly, the immobilized GOD in the biE-HNFs-beads oxidized Glu to produce H₂O₂ in the presence of O₂. Secondly, the immobilized reduction state HRP(red) in biE-HNFs-beads was converted to the oxidation state HRP(ox) by H₂O₂ (Ariza-Avidad et al., 2016). Thirdly, the mediator ABTS(red) was oxidized by HRP(ox) to form the radical ABTS⁺(ox) with higher redox potential. Finally, ABTS⁺ reacted non-enzymatically with acridine to reduce ABTS⁺ to the initial state of ABTS and oxidized acridine to complete the entire catalytic cycle of biE-HNFs-beads (Xue et al., 2020). This eventually led to the complete degradation of acridine molecules. In this process, the redox mediator ABTS not only played an electron-mediated role between catalyst and substrate, but also enhanced the catalytic ability of HRP and broadened the substrate range of HRP.

3.2

3.2 Characterisation of biE-HNFs-beads

SEM, TEM and EDS were used to characterize the structure of the biE-HNFs-beads. Low-resolution SEM image (Fig. 2A) presented that biE-HNFs-beads were regular spheres with a diameter of about 3 mm, which was conducive to separating from the reaction system during operation. In the high-resolution SEM image (Fig. 2B), many nanoflowers (average size, ∼ 3 μm) assembled from nanosheets grow on the surface of biE-HNFs-beads, which significantly increased the surface-to-volume ratios of the biE-HNFs-beads and enabled them to load more bienzyme molecules.

Low-resolution SEM image (A), high-resolution SEM image (B) of biE-HNFs-beads. TEM image (C, D) of nanoflower attached on the surface of biE-HNFs-beads. EDS image (E) of nanoflower attached on the surface of biE-HNFs-beads and the presence of main elements (F − L) in this region.

The TEM image demonstrated that the biE-HNFs-beads were assembled by many small copper phosphate crystals and biE-HNFs (Fig. 2C). In addition, the edge structure of the biE-HNFs can be clearly shown in Fig. 2D (Li et al., 2014). Furthermore, the presence of different elements in biE-HNFs-beads could be determined by EDS analysis. As shown in Fig. 2E − 2L, Cu, O, P, C, N, Fe and Ca elements were found in biE-HNFs-beads (Gul and Ocsoy, 2021). Among them, Cu, O and P elements were all attributed to Cu₃(PO₄)₂. C and N elements were derived from Ce-Cs beads, Fe element just came from HRP, and Ca element only went out of GOD. This indicated that the biE-HNFs had been successfully adhered to the surface of the beads.

In order to further verify the successful synthesis of biE-HNFs-beads, FT-IR spectroscopy was applied to characterize the molecular structure of biE-HNFs-beads, biE-HNFs powder, Cu₃(PO₄)₂ crystal and Ce-Cs/@DA beads. The assignment of the observed bands or peaks were discussed as below according to the literatures. As displayed in Fig. 3A, the same characteristic peaks appeared at 1049, 990, 630 and 557 cm⁻¹ for Cu₃(PO₄)₂ crystal, biE-HNFs powder and biE-HNFs-beads, respectively, which was derived from the vibration of PO₄³⁻ (Ren et al., 2018). Besides, the diffraction peaks of biE-HNFs-beads and biE-HNFs powder at around 1600, 1540, 2850, 2910 and 3000 cm⁻¹ assigned to the amide carbonyl group in HRP-GOD bienzyme molecule (Wu et al., 2022). It was worth noting that the biE-HNFs-beads and Ce-Cs/@DA beads had similar characteristic diffraction peaks at 1334 and 1240 cm⁻¹, which was caused by dopamine modification (Liu et al., 2021). Both results demonstrated that the biE-HNFs were successfully attached to the surface of the beads.

FT-IR spectra (A): Cu3(PO4)2 (a), biE-HNFs (b), biE-HNFs-beads (c) and Ce-Cs@DA beads (d). XRD patterns (B, C): Cu3(PO4)2 (a), biE-HNFs (b), biE-HNFs-beads (c) and Ce-Cs@DA beads (d). EPR spectra (D, E): biE-HNFs-beads + ABTS + Glu (a), biE-HNFs + ABTS + Glu (b) and free HRP-GOD + ABTS + Glu (c). Michaelis-Menten parameters (F) of free HRP-GOD (a) and biE-HNFs-beads (b).

The structures of biE-HNFs-beads, biE-HNFs, Cu₃(PO₄)₂ crystal and Ce-Cs beads were analyzed by XRD. According to Fig. 3B, XRD patterns of Cu₃(PO₄)₂ crystal and biE-HNFs powder were in good agreement with the position of diffraction peaks and relative intensities, indicating that the biE-HNFs powder was mainly composed of Cu₃(PO₄)₂. From Fig. 3C, the three characteristic peaks of biE-HNFs-beads at 8°, 12° and 30° were consistent with the crystal structure of Cu₃(PO₄)₂ (Yu et al., 2015), while the peak patterns at 15° and 22° corresponded to the characteristic peaks of Ce-Cs beads (Gu et al., 2022), suggesting the successful combination of the biE-HNFs-beads.

EPR could capture radicals in the reaction system. The samples, biE-HNFs-beads + ABTS + Glu, biE-HNFs + ABTS + Glu, as well as free HRP-GOD + ABTS + Glu, were designed to verify the reaction cascade process and the electron transfer of intermediate products by EPR, respectively. ABTS (30 mg) and Glu (20 mg) were added into PBS buffer solution (10 mL, pH 7.0), and the reaction was initiated by the addition of biE-HNFs-beads (1 g), biE-HNFs powder (1 mg) and free HRP-GOD (10 mg) as the last component. After 30 min, the electron transfer radical signal was monitored by EPR. From the EPR spectrum, it could be observed that ABTS radical signal (Fig. 3D, special peaks are marked with *) and hydroxyl radical signal (Fig. 3E, special peaks are marked with *) exist in all three systems, which indicated that the bienzyme was successfully immobilized on the biE-HNFs and the biE-HNFs-beads. The system existed in two kinds of reactions, the first reaction generated hydroxyl free radicals (Ma, N., Xue, P. et al., 2021), the second reaction built on the first reaction to generate ABTS radicals (Huang et al., 2022). However, the signal of ABTS radical in PBS buffer solution was weaker, and the signal of hydroxyl radical was more potent than that of free bienzyme and biE-HNFs powder when the biE-HNFs-beads were used to initiate the reaction. It may be attributed to the large specific surface area of biE-HNFs powder and the small mass transfer resistance between the powder and solution, which accelerated the electron transfer speed in reaction process. Meanwhile, although the biE-HNFs-beads had a large specific surface area, the mass transfer resistance of the carrier limits the electron transfer speed in the reaction process to a certain extent. The specific surface area of biE-HNFs powders was determined by BET method (Supporting Information Fig. S1 and S2). The results showed that BET surface area of biE-HNFs was 24.4573 m²/g, which was higher than the results reported in previous literatures (Chung et al., 2018; Wu et al., 2022). The charge transport capacity of biE-HNFs was investigated by EIS (Supporting Information Fig. S3). The radius of the semicircle indicates the charge transfer resistance (R_ct). The smaller the R_ct value, the lower the charge transfer resistance and the higher the reaction kinetics. As can be seen from the Fig. S3, the radius of biE-HNFs was smaller than that of bare electrodes, indicating that they had lower mass transfer resistance and faster reaction kinetics (Arif et al., 2018; Fang et al., 2022).

3.3

3.3 The kinetics of biE-HNFs-beads

The kinetic constants, the maximum reaction rate (V_max) and apparent Michaelis–Menten constant (K_m), of free bienzyme and biE-HNFs-beads were calculated by measuring their catalytic rates at different concentrations of ABTS. The lower the value of K_m, the higher is the affinity of the enzyme to the substrate. The lesser the value of K_m, the higher is the affinity for the substrate. As shown in Fig. 3F and Table 1, the K_m value for biE-HNFs-beads (4.12 mmol/L) was found to be 3-fold more than that of the free bienzyme (1.36 mmol/L) demonstrating a lower affinity for the substrate caused by diffusional limitations. Nevertheless, the V_max of biE-HNFs-beads (0.48 mmol·min/L) was higher than that of free bienzyme (0.07 mmol·min/L). On the one hand, it is because after the immobilization of bienzyme, the activity of enzyme is improved in the reaction microenvironment and the catalytic rate is accelerated. On the other hand, the adsorption of the carrier increases the local concentration and speeds up the reaction. This phenomenon is consistent with some literature reports. For example, Sun’s group (Sun et al., 2016) encapsulated laccase in chitosan hydrogel grafted with polyacrylamide and measured its reaction kinetics. It was also found that the affinity between immobilized enzyme and substrate was significantly reduced, but the reaction rate between free enzyme and substrate was nearly 1.8 times higher than that of free enzyme.

Table 1 Kinetic parameters of Michaelis–Menten constants of free bienzyme and biE-HNFs-beads.

biocatalyst	Michaelis-Menten parameters
biocatalyst	K_m (mmol/L)	V_max (mmol·min/L)
Free bienzyme	1.36	0.07
biE-HNFs-beads	4.12	0.48

3.4

3.4 The optimization of formation conditions for biE-HNFs-beads

3.4.1

3.4.1 The effect of synthesis temperature

The effects of synthesis temperature on enzyme activity and bienzyme encapsulation yield of the biE-HNFs-beads were studied under the condition of controlling other synthesis conditions. As shown in Fig. 4A, the enzyme activity and the bienzyme encapsulation yield of the biE-HNFs-beads synthesized at 0 °C reached the maximum, and they decreased gradually with the increase in temperature. The reason for this phenomenon is that, on the one hand, with the decrease in temperature, the dispersity of the enzyme becomes worse, resulting in the increased concentration of enzyme around the nucleation site. On the other hand, the increase in temperature may cause the conformational change of the enzymes, thus affecting the enzyme activity. Similar conclusions have been reported in the literature (Li et al., 2016b).

Effect of synthesis temperature (A), concentration ratios of HRP:GOD in solution (B), bienzyme concentrations (C) and different preparation methods on the synthesis of the beads-supported nanoflowers biocatalyst (D): GOD@HRP-HNFs-beads (a), HRP@GOD-HNFs-beads (b), biE-HNFs-beads (c), HRP-HNFs-beads (d) and GOD-HNFs-beads (e).

3.4.2

3.4.2 The effect of the concentration ratios of HRP:GOD in solution

The influences of the concentration ratios of HRP:GOD in solution on enzyme activity and bienzyme encapsulation yield of biE-HNFs-beads were investigated under the condition that the total enzyme mass was the same. As can be seen from Fig. 4B, when the concentration ratio of HRP:GOD in solution was 2:1, both enzyme activity and bienzyme encapsulation yield of biE-HNFs-beads were the highest. There are two reasons for this phenomenon. For one thing, when GOD is small, the generated H₂O₂ cannot meet the needs of HRP enzymatic reaction. For another, when GOD is enormous, too much H₂O₂ will lead to HRP deactivation, and the reduction of HRP will further reduce the catalytic efficiency. Therefore, we selected HRP:GOD was 2:1 as the bienzyme concentration ratios for preparing biE-HNFs-beads in the following experiments.

3.4.3

3.4.3 The effect of bienzyme concentration

The effects of bienzyme concentration on enzyme activity and bienzyme encapsulation yield of the biE-HNFs-beads were studied when other synthesis conditions were not changed. As shown in Fig. 4C, with increase of bienzyme concentration, the enzyme activity and the bienzyme encapsulation yield of the biE-HNFs-beads increased first and then decreased. The reason may be the structural differences of biE-HNFs-beads when the concentration of bienzyme changes. In the beginning, when the bienzyme concentration increased from 0.5 mg/mL to 1.0 mg/mL, the more nanoflowers loaded on the surface of biE-HNFs-beads, the larger the specific surface area, and the higher enzyme activity and bienzyme encapsulation yield. However, when the concentration larger than1.0 mg/mL, the nanoflowers supported on the surface of biE-HNFs-beads grew further, the flower-like structure was agglomerated, and the specific surface area was greatly reduced. As a result, the enzyme activity and the bienzyme encapsulation yield decreased gradually.

3.4.4

3.4.4 The effect of different preparation methods

Under the control of the total enzyme mass and the corresponding HRP-GOD mass ratio, the influences of different catalyst preparation methods on the formation of the beads-supported nanoflowers biocatalyst were investigated. Fig. 4D shows the activity and bienzyme encapsulation yield of biE-HNFs-beads, HRP@GOD-HNFs-beads, GOD@HRP-HNFs-beads, HRP-HNFs-beads and GOD-HNFs-beads. Apparently, although all of the catalysts exhibited high activity and bienzyme encapsulation yield, however, compared with the immobilized bienzyme prepared by the fractional method for GOD@HRP-HNFs-beads (Fig. 4D, columns a) and HRP@GOD-HNFs-beads (columns b), one-pot method for preparing biE-HNFs-beads (column c) showed better activity and encapsulation yield. This might be related to the spatial distribution of the bienzyme that formed nanoflowers (Han et al.0.2020), we speculated that the spatial distance between HRP and GOD in biE-HNFs-beads also might be shorter, thus the intermediate H₂O₂ could be rapidly transmitted from the active site of GOD to the active site of HRP. This distribution was more consistent with the cascade reaction sequence in the catalytic process. In addition, compared with the immobilized single enzyme HRP-HNFs-beads (Fig. 4D, columns d) and GOD-HNFs-beads (columns e), the immobilized bienzyme biE-HNFs beads (column c) showed better activity and encapsulation yield. This might be because the synergistic effect of HRP and GOD could avoid the accumulation of some harmful intermediates by generating and consuming intermediates in situ, and thus improving the stability of the enzymes and accelerating the reaction rate (Han et al., 2019; Zhu et al., 2017). This result was also elaborated in our previous work (Gu et al, 2022). Therefore, the immobilized bienzyme biE-HNFs-beads prepared by the one-pot method were selected as the subsequent research object (Li et al., 2014).

The optimum synthesis conditions were determined by discussing the effects of temperature, bienzyme ratio, bienzyme concentration, synthesis method on enzyme activity and encapsulation yield after biocatalyst formation to ensure that the biocatalyst production process with less input to obtain a greater return. These data can provide a basis for future industrial production and wastewater treatment.

3.5

3.5 Properties of biE-HNFs-beads

3.5.1

3.5.1 Effect of reaction parameters on the catalytic degradation of acridine

High concentrations of acridine may adversely affect the activity of the bienzyme. The effect of concentration on acridine degradation was studied using the biE-HNFs-beads. As could be observed in Fig. 5A, the degradation rate of acridine remained above 99.0 % as the concentration of acridine increased from 5 mg/L to 15 mg/L. Nevertheless, when the concentration of acridine increased from 15 mg/L to 45 mg/L, the degradation rate of acridine decreased gradually. This might be due to the biE-HNFs-beads undergoing the substrate inhibition in high concentration of acridine (Sun et al., 2017).

Effects of various reaction parameters on the catalytic degradation of acridine: acridine concentration (A), ABTS dosage (B), glucose dosage (C) and biocatalyst dosage (D).

The redox medium ABTS can significantly improve the degradation rate of acridine by HRP. The biE-HNFs-beads were used to study the effect of ABTS dosage on acridine degradation. As seen from Fig. 5B, with the increase of ABTS dosage from 1 mg/mL to 3 mg/mL, the degradation rate of acridine gradually increased to 99.0 % and then tended to level off. This indicated that 3 mg/mL ABTS molecule could meet the demand of acridine degradation reaction, and excessive ABTS did not significantly promote the degradation efficiency (Huang et al., 2022).

The addition of glucose is a key step in the acridine degradation reaction. Without glucose, the reaction cannot proceed. The influence of glucose dosage on acridine degradation was examined by the biE-HNFs-beads. As shown in Fig. 5C, the degradation of acridine first increased and then remained unchanged with the increase in glucose dosage, suggesting that the optimal dosage of glucose used in the acridine degradation reaction was 2 mg/mL (Yang et al., 2014).

The dosage of the biocatalyst determines the degradation rate of acridine to a large extent. Fig. 5D shows the effect of the biE-HNFs-beads dosage on acridine degradation. Clearly, the degradation rate of acridine was significantly enhanced when the dosage of biE-HNFs-beads was increased from 0.02 g/mL to 0.12 g/mL. However, when the dosage of biE-HNFs-beads was higher than 0.10 g/L, the increasing trend of acridine degradation rate was not noticeable. Therefore, the optimal dosage of biE-HNFs-beads was 0.10 g/L (Han et al., 2019).

In this part, the influence of different reaction parameters on the degradation efficiency of acridine was discussed to determine the optimal conditions of the biocatalyst, so as to ensure the maximum efficiency of the biocatalyst to degrade pollutants.

3.5.2

3.5.2 Stability study of biE-HNFs-beads

In order to investigate the temperature resistance of the biocatalyst, free HRP-GOD, biE-HNFs and biE-HNFs-beads were used to react in acridine solution at different temperatures (20 °C ∼ 60 °C) for 8 h. As demonstrated in Fig. 6A, free HRP-GOD (curve a), biE-HNFs (curve b) and biE-HNFs-beads (curve c) had similar curves. They all showed the best degradation efficiency for acridine at 30 °C, and degradation efficiency decreased when the temperature was too high or too low. Nevertheless, compared with free HRP-GOD and biE-HNFs, biE-HNFs-beads maintained a higher degradation rate of acridine in the tested temperature range, showing a more robust temperature resistance stability. Similarly, to determine the pH resistance of the biocatalyst, free HRP-GOD, biE-HNFs and biE-HNFs-beads were used to degrade acridine solutions of different pH values for 8 h, respectively. As shown in Fig. 6B, free HRP-GOD (curve a), biE-HNFs (curve b) and biE-HNFs-beads (curve c) all showed the best degradation efficiency for acridine at pH 7.0, and the degradation efficiency decreased at extreme pH conditions. However, compared with free HRP-GOD and biE-HNFs, biE-HNFs-beads had higher degradation rate of acridine in the test pH range, showing stronger pH resistance stability. It could be interpreted as the result of the extreme temperature and pH would destroy the structure of the bienzyme, so the degradation rate of free bienzyme to acridine was significantly reduced. However, after the immobilization of bienzyme, the protective effect of the carrier on the bienzyme reduced the occurrence of drastic conformational changes of bienzyme molecules under extreme conditions, which preserved the bienzyme activity to a certain extent (Wu et al., 2022). Therefore, compared with the free HRP-GOD, biE-HNFs and biE-HNFs-beads had better temperature and pH resistance stability. Nevertheless, because the biE-HNFs-beads further improved the mechanical strength and stability of biE-HNFs, it showed better temperature and pH resistance stability than biE-HNFs.

The thermal stability (A): biE-HNFs-beads (a), biE-HNFs (b) and free HRP-GOD (c), pH stability (B): biE-HNFs-beads (a), biE-HNFs (b) and free HRP-GOD (c). The reusability (C) and storage stability (D) of the biocatalyst for acridine degradation, and the activity recovery of biE-HNFs-beads.

One of the unique characteristics of immobilized enzyme is that they can be recycled (Feng et al., 2021; Feng et al., 2022). Therefore, to effectively reduce the cost, the reusability of the immobilized enzyme has always been a concern in industrial applications. In this experiment, the reusability of biE-HNFs-beads and biE-HNFs and the active recovery rate of biE-HNFs-beads were investigated by performing 10 consecutive acridine degradation cycles under the same operating conditions. Acridine solution prepared freshly (10 mL, 15 mg/L) was added each cycle, and another degradation was carried out under optimal conditions. The biE-HNFs-beads could be recovered by simple filtration at the end of each cycle because of the larger size, while the biE-HNFs needs to be separated from the reaction system through complex centrifugation steps due to their tiny structure. Fig. 6C displays the recycling performance of biE-HNFs-beads and biE-HNFs and the active recovery rate of biE-HNFs-beads in 10 successive operations. After 4 cycles, the degradation rate of acridine by biE-HNFs was reduced to 66.4 %, lower than that of the biE-HNFs-beads (90.2 %). At the end of 10 degradation cycles, the acridine degradation rate and activity recovery of biE-HNFs-beads to acridine was still up to 60.0 %. This reusability of the biE-HNFs-beads facilitated the continuous use of immobilized bienzyme in industrial applications. The reason for the decreased in enzyme activity might be related to the deactivation of HRP-GOD caused by the denaturation of the enzymes and the leakage of enzymes from the beads (Altinkaynak et al., 2017).

The storage stability of immobilized enzyme is a necessary condition for the application of immobilized enzyme and an essential factor affecting their practical application (Cui et al., 2018a). Therefore, the stability of biE-HNFs-beads, biE-HNFs and free bienzyme and the recovery of activity of biE-HNFs-beads were compared in this experiment. The biE-HNFs-beads, biE-HNFs and free bienzyme were immersed in PBS solution at 4 °C for 2 months, respectively, and then appropriate amounts were removed every 10 days for degradation of acridine. Results as shown in Fig. 6D, the activity recovery of biE-HNFs-beads after 60 days of storage was more than 80%, and the degradation rate of acridine was 82.2 %, far better than that of biE-HNFs (60.0 %) and free bienzyme (40.0 %). Obviously, biE-HNFs beads presented better storage stability than biE-HNFs and free bienzyme. This was attributed to the structure of biE-HNFs-beads restricting and protecting the conformation of the enzyme, which reduced the degeneration and autolysis of the enzymes and improved the storage stability of the enzymes to a certain extent (Kiani et al., 2022).

3.6

3.6 Possible degradation products and pathways of acridine

In the biE-HNFs-beads degradation experiment, acridine was wholly degraded within 8 h, six products of acridine were identified by LC-MS. The detailed information of degradation products was proposed in Fig. 7A and 7B, which were named a (m/z = 325.11), b (m/z = 74.06), c (m/z = 111.09), d (m/z = 83.06), e (m/z = 360.15) and f (m/z = 515.02), respectively. Based on the identification of degradation products, four possible degradation pathways of acridine were proposed (Fig. 7C). Among them, the products a, b, c, e and f were produced by the stepwise cleavage of the benzene ring on the acridine molecule, and d was generated by the polymerization of b and c. In conclusion, the biE-HNFs beads could completely degrade acridine in wastewater into small molecular compounds.

Mass spectrum of LC-MS after the complete degradation of acridine (A, B). Four possible routes (I, II, III, IV) and six possible products (a ∼ f) of acridine degradation in biE-HNFs-beads (C).

4 Conclusions

The biocatalyst with the multi-layered flower-like surface structure was successfully prepared by combining the bienzyme-inorganic hybrid nanoflowers with the traditional beads carries through the adhesion of polydopamine. The unique and distinguishing characteristics of the system are as follows: (i) Both HRP and GOD were used as organic components to synthesize biE-HNFs in this experiment, so that the biocatalyst could carry out a bienzyme cascade reaction in a single system. Since the bienzyme components in a single nanoflower were very close to each other, and the intermediates were not separated, this novel biocatalyst greatly reduced the diffusion and decomposition of H₂O₂, significantly improved the stability of the bienzyme, and accelerated the degradation of pollutants. (ii) The experiment innovatively combined biE-HNFs structure and beads structure, which not only solved the problems of poor stability, difficult recovery and poor reusability of biE-HNFs structure, but also solved the problems of low enzyme load and high mass transfer resistance of beads structure. (iii) The biocatalyst could degrade acridine in wastewater with green and safe degradation process, and the degradation rate of acridine could reach 99.9 %. Besides, LC-MS analysis confirmed that acridine was degraded and converted to less toxic metabolites. Compared with free bienzyme and the biE-HNFs, the biE-HNFs-beads had better stability, reusability and storage stability. In general, the construction of supporter-supported nanoflowers biocatalyst provided a new method for improving the mechanical strength, stability and reusability of immobilized enzymes, which was expected to be widely used in the field of wastewater treatment.

CRediT authorship contribution statement

Yaohua Gu: Conceptualization, Formal analysis, Writing – review & editing, Funding acquisition. Lin Yuan: Conceptualization, Formal analysis, Investigation, Data curation, Writing – original draft, Visualization. Mingming Li: Conceptualization, Investigation, Data curation, Writing – original draft, Visualization. Ying Liu: Data curation, Visualization. Xiaoyan Bai: Data curation, Visualization. Keren Shi: Conceptualization, Formal analysis, Writing – review & editing, Funding acquisition. Huiqin Yao: Conceptualization, Formal analysis, Writing – review & editing, Funding acquisition.

Acknowledgments

This work was financially supported by the Key Research and Development Projects of Ningxia Province of China (2021BEB04055), Natural Science Foundation of Ningxia (2022AAC03146, 2021AAC03056), CAS “Light of West China Program” of China (XAB2020YW16) and School-level Special Talents Launching Project of Ningxia Medical University (XT2020017).

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References

Abdelhameed R.M., Emam H.E., . Design of ZIF(Co & Zn)@wool composite for efficient removal of pharmaceutical intermediate from wastewater. J. Colloid Interface Sci.. 2019;552:494-505.
[CrossRef] [Google Scholar]
Altinkaynak, C., ÖZdemİR, N., ÖÇSoy, İ., 2021. A rational synthesis of magnetic nanoparticles incorporated horseradish peroxidase nanoflower and its use for the removal of phenol through oxidative coupling reaction with great reusability. Mugla. J. Sci. Techn. 7(2), 59−66. https://doi.org/10.22531/muglajsci.982993.
Altinkaynak C., Tavlasoglu S., Kalin R., Sadeghian N., Ozdemir H., Ocsoy I., Ozdemir N., . A hierarchical assembly of flower-like hybrid Turkish black radish peroxidase-Cu²⁺ nanobiocatalyst and its effective use in dye decolorization. Chemosphere. 2017;182:122-128.
[CrossRef] [Google Scholar]
Arif M., Yasin G., Shakeel M., Fang X., Gao R., Ji S., Yan D., . Coupling of bifunctional comn-layered double hydroxide@graphitic C₃N₄ nanohybrids towards efficient photoelectrochemical overall water splitting. Chem. Asian J.. 2018;13:1045-1052.
[CrossRef] [Google Scholar]
Ariza-Avidad M., Salinas-Castillo A., Capitan-Vallvey L.F., . A 3D μPAD based on a multi-enzyme organic-inorganic hybrid nanoflower reactor. Biosens. Bioelectron.. 2016;77:51-55.
[CrossRef] [Google Scholar]
Chung M., Nguyen T.L., Tran T.Q.N., Yoon H.H., Kim I.T., Kim M.I., . Ultrarapid sonochemical synthesis of enzyme-incorporated copper nanoflowers and their application to mediatorless glucose biofuel cell. Appl. Surf. Sci.. 2018;429:203-209.
[CrossRef] [Google Scholar]
Cui J., Feng Y., Jia S., . Silica encapsulated catalase@metal-organic framework composite: A highly stable and recyclable biocatalyst. Chem. Eng. J.. 2018;351:506-514.
[CrossRef] [Google Scholar]
Cui J., Jia S., . Organic–inorganic hybrid nanoflowers: a novel host platform for immobilizing biomolecules. Coord. Chem. Rev.. 2017;352:249-263.
[CrossRef] [Google Scholar]
Cui J., Zhao Y., Liu R., Zhong C., Jia S., . Surfactant-activated lipase hybrid nanoflowers with enhanced enzymatic performance. Sci. Rep.. 2016;6:27928.
[CrossRef] [Google Scholar]
Cui J., Ren S., Lin T., Feng Y., Jia S., . Shielding effects of Fe³⁺-tannic acid nanocoatings for immobilized enzyme on magnetic Fe₃O₄@silica core shell nanosphere. Chem. Eng. J.. 2018;343:629-637.
[CrossRef] [Google Scholar]
Du Y., Jia X., Zhong L., Jiao Y., Zhang Z., Wang Z., Feng Y., Bilal M., Cui J., Jia S., . Metal-organic frameworks with different dimensionalities: an ideal host platform for enzyme@MOF composites. Coord. Chem. Rev.. 2022;454:214327
[CrossRef] [Google Scholar]
Fang X., Tang Y., Ma Y.-J., Xiao G., Li P., Yan D., . Ultralong-lived triplet excitons of room-temperature phosphorescent carbon dots located on g-C₃N₄ to boost photocatalysis. Sci. China Mater.. 2022;66:664-671.
[CrossRef] [Google Scholar]
Feng Y., Hu H., Wang Z., Du Y., Zhong L., Zhang C., Jiang Y., Jia S., Cui J., . Three-dimensional ordered magnetic macroporous metal-organic frameworks for enzyme immobilization. J. Colloid. Interf. Sci.. 2021;590:436-445.
[CrossRef] [Google Scholar]
Feng Y., Du Y., Kuang G., Zhong L., Hu H., Jia S., Cui J., . Hierarchical micro- and mesoporous ZIF-8 with core–shell superstructures using colloidal metal sulfates as soft templates for enzyme immobilization. J. Colloid. Interf. Sci.. 2022;610:709-718.
[CrossRef] [Google Scholar]
Fu M., Xing J., Ge Z., . Preparation of laccase-loaded magnetic nanoflowers and their recycling for efficient degradation of bisphenol A. Sci. Total. Environ.. 2019;651:2857-2865.
[CrossRef] [Google Scholar]
Ge J., Lei J., Zare R.N., . Protein-inorganic hybrid nanoflowers. Nat. Nanotechnol.. 2012;7:428-432.
[CrossRef] [Google Scholar]
Gu Y., Yuan L., Li M., Wang X., Rao D., Bai X., Shi K., Xu H., Hou S., Yao H., . Co-immobilized bienzyme of horseradish peroxidase and glucose oxidase on dopamine-modified cellulose–chitosan composite beads as a high-efficiency biocatalyst for degradation of acridine. RSC Adv.. 2022;11:23006-23016.
[CrossRef] [Google Scholar]
Gul O.T., Ocsoy I., . Co-Enzymes based nanoflowers incorporated-magnetic carbon nanotubes: a new generation nanocatalyst for superior removal of cationic and anionic dyes with great repeated use. Environ. Technol. Inno.. 2021;24:101992
[CrossRef] [Google Scholar]
Han J., Luo P., Wang L., Li C., Mao Y., Wang Y., . Construction of magnetic nanoflower biocatalytic system with enhanced enzymatic performance by biomineralization and its application for bisphenol A removal. J. Hazard. Mater.. 2019;380:120901
[CrossRef] [Google Scholar]
Han J., Luo P., Wang L., Wu J., Li C., Wang Y., . Construction of a multienzymatic cascade reaction system of coimmobilized hybrid nanoflowers for efficient conversion of starch into gluconic acid. ACS Appl. Mater. Inter.. 2020;12:15023-15033.
[CrossRef] [Google Scholar]
Hiner A.N., Hernandez-Ruiz J., Rodriguez-Lopez J.N., Arnao M.B., Varon R., Garcia-Canovas F., Acosta M., . The inactivation of horseradish peroxidase isoenzyme A2 by hydrogen peroxide: an example of partial resistance due to the formation of a stable enzyme intermediate. J. Biol. Inorg. Chem.. 2001;6:504-516.
[CrossRef] [Google Scholar]
Huang Y., Ran X., Lin Y., Ren J., Qu X., . Self-assembly of an organic-inorganic hybrid nanoflower as an efficient biomimetic catalyst for self-activated tandem reactions. Chem. Commun.. 2015;51:4386-4389.
[CrossRef] [Google Scholar]
Huang Y., Zou J., Lin J., Yang H., Wang M., Li J., Cao W., Yuan B., Ma J., . ABTS as both activator and electron shuttle to activate persulfate for diclofenac degradation: formation and contributions of ABTS+, SO4−, and OH. Environ. Sci. Technol. 2022
[CrossRef] [Google Scholar]
Jiang Y., Zhai J., Zhou L., He Y., Ma L., Gao J., . Enzyme@silica hybrid nanoflowers shielding in polydopamine layer for the improvement of enzyme stability. Biochem. Eng. J.. 2018;132:196-205.
[CrossRef] [Google Scholar]
Kaiser J., Feng Y., Bollag J., . Microbial metabolism of pyridine, quinoline, acridine, and their derivatives under aerobic and anaerobic conditions. Microbiol. Rev.. 1996;60(3):483-498.
[CrossRef] [Google Scholar]
Kiani M., Mojtabavi S., Jafari-Nodoushan H., Tabib S.R., Hassannejad N., Faramarzi M.A., . Fast anisotropic growth of the biomineralized zinc phosphate nanocrystals for a facile and instant construction of laccase@Zn₃(PO₄)₂ hybrid nanoflowers. Int. J. Biol. Macromol.. 2022;204:520-531.
[CrossRef] [Google Scholar]
Lee H.R., Chung M., Kim M.I., Ha S.H., . Preparation of glutaraldehyde-treated lipase-inorganic hybrid nanoflowers and their catalytic performance as immobilized enzymes. Enzyme Microb. Technol.. 2017;105:24-29.
[CrossRef] [Google Scholar]
Li Y., Fei X., Liang L., Tian J., Xu L., Wang X., Wang Y., . The influence of synthesis conditions on enzymatic activity of enzyme-inorganic hybrid nanoflowers. J. Mol. Catal. B: Enzym.. 2016;133:92-97.
[CrossRef] [Google Scholar]
Li H., Hou J., Duan L., Ji C., Zhang Y., Chen V., . Graphene oxide-enzyme hybrid nanoflowers for efficient water soluble dye removal. J. Hazard. Mater.. 2017;338:93-101.
[CrossRef] [Google Scholar]
Li M., Luo M., Li F., Wang W., Liu K., Liu Q., Wang Y., Lu Z., Wang D., . Biomimetic copper-based inorganic–protein nanoflower assembly constructed on the nanoscale fibrous membrane with enhanced stability and durability. J. Phys. Chem. C. 2016;120:17348-17356.
[CrossRef] [Google Scholar]
Li K., Wang J., He Y., Abdulrazaq M.A., Yan Y., . Carbon nanotube-lipase hybrid nanoflowers with enhanced enzyme activity and enantioselectivity. J. Biotechnol.. 2018;281:87-98.
[CrossRef] [Google Scholar]
Li Y., Xie G., Qiu J., Zhou D., Gou D., Tao Y., Li Y., Chen H., . A new biosensor based on the recognition of phages and the signal amplification of organic-inorganic hybrid nanoflowers for discriminating and quantitating live pathogenic bacteria in urine. Sensors Actuators B: Chem.. 2018;258:803-812.
[CrossRef] [Google Scholar]
Li Z., Zhang Y., Su Y., Ouyang P., Ge J., Liu Z., . Spatial co-localization of multi-enzymes by inorganic nanocrystal-protein complexes. Chem. Commun.. 2014;50:12465-12468.
[CrossRef] [Google Scholar]
Li P., Zhang T., Mushtaq M.A., Wu S., Xiang X., Yan D., . Research progress in organic synthesis by means of photoelectrocatalysis. Chem. Rec.. 2021;21:841-857.
[CrossRef] [Google Scholar]
Li C., Zhao J., Zhang Z., Jiang Y., Bilal M., Jiang Y., Jia S., Cui J., . Self-assembly of activated lipase hybrid nanoflowers with superior activity and enhanced stability. Biochem. Eng. J.. 2020;158:107582
[CrossRef] [Google Scholar]
Liu X., Xue P., Jia F., Shi K., Gu Y., Ma L., Li R., . A novel approach to efficient degradation of indole using co-immobilized horseradish peroxidase-syringaldehyde as biocatalyst. Chemosphere. 2021;262:128411
[CrossRef] [Google Scholar]
Ma, N., Xue, P., Li R., Ma L., Shi K., . Flower-like hybrid composite MoS₂/NH₂-MIL-101(Cr): a highly efficient photocatalyst for degrading indole under visible light. J. Mater. Res.. 2021;36:4460-4474.
[CrossRef] [Google Scholar]
Mohammad M., Ahmadpoor F., Shojaosadati S.A., . Mussel-inspired magnetic nanoflowers as an effective nanozyme and antimicrobial agent for biosensing and catalytic reduction of organic dyes. ACS Omega. 2020;5:18766-18777.
[CrossRef] [Google Scholar]
Mushtaq M.A., Kumar A., Yasin G., Arif M., Tabish M., Ibraheem S., Cai X., Ye W., Fang X., Saad A., Zhao J., Ji S., Yan D., . 3D interconnected porous Mo-doped WO₃@CdS hierarchical hollow heterostructures for efficient photoelectrochemical nitrogen reduction to ammonia. Appl. Catal. B-Environ.. 2022;317:121711
[CrossRef] [Google Scholar]
Ocsoy I., Dogru E., Usta S., . A new generation of flowerlike horseradish peroxides as a nanobiocatalyst for superior enzymatic activity. Enzyme. Microb. Technol.. 2015;75–76:25-29.
[CrossRef] [Google Scholar]
Pitzalis F., Monduzzi M., Salis A., . A bienzymatic biocatalyst constituted by glucose oxidase and Horseradish peroxidase immobilized on ordered mesoporous silica. Micropor. Mesopor. Mater.. 2017;241:145-154.
[CrossRef] [Google Scholar]
Razzaghi M., Homaei A., Vianello F., Azad T., Sharma T., Nadda A.K., Stevanato R., Bilal M., Iqbal H.M.N., . Industrial applications of immobilized nano-biocatalysts. Bioprocess. Biosyst. Eng.. 2022;45:237-256.
[CrossRef] [Google Scholar]
Ren W., Fei X., Tian J., Li Y., Jing M., Fang H., Xu L., Wang Y., . Multiscale immobilized lipase for rapid separation and continuous catalysis. New J. Chem.. 2018;42:13471-13478.
[CrossRef] [Google Scholar]
Ren W., Li Y., Wang J., Li L., Xu L., Wu Y., Wang Y., Fei X., Tian J., . Synthesis of magnetic nanoflower immobilized lipase and its continuous catalytic application. New J. Chem.. 2019;43:11082-11090.
[CrossRef] [Google Scholar]
Rong J., Zhang T., Qiu F., Zhu Y., . Preparation of efficient, stable, and reusable laccase–Cu₃(PO₄)₂ hybrid microspheres based on copper foil for decoloration of congo red. ACS Sustain. Chem. Eng.. 2017;5:4468-4477.
[CrossRef] [Google Scholar]
Sharma N., Parhizkar M., Cong W., Mateti S., Kirkland M.A., Puri M., Sutti A., . Metal ion type significantly affects the morphology but not the activity of lipase–metal–phosphate nanoflowers. RSC Adv.. 2017;7:25437-25443.
[CrossRef] [Google Scholar]
Sun H., Huang W., Yang H., . Co-immobilization of laccase and mediator through a self-initiated one-pot process for enhanced conversion of malachite green. J. Colloid Interf. Sci.. 2016;471:20-28.
[CrossRef] [Google Scholar]
Sun K., Huang Q., Li S., . Transformation and toxicity evaluation of tetracycline in humic acid solution by laccase coupled with 1-hydroxybenzotriazole. J. Hazard. Mater.. 2017;331:182-188.
[CrossRef] [Google Scholar]
Wang C., Hu B., Wang J., Gao J., Li G., Xiong W.W., Zou B., Suzuki M., Aratani N., Yamada H., Huo F., Lee P.S., Zhang Q., . Rewritable multilevel memory performance of a tetraazatetracene donor-acceptor derivative with good endurance. Chem. Asian J.. 2015;10:116-119.
[CrossRef] [Google Scholar]
Wen H., Zhang L., Du Y., Wang Z., Jiang Y., Bian H., Cui J., Jia S., . Bimetal based inorganic-carbonic anhydrase hybrid hydrogel membrane for CO₂ capture. J. CO₂ Util.. 2020;39:101171
[CrossRef] [Google Scholar]
Wu J., Ma X., Li C., Zhou X., Han J., Wang L., Dong H., Wang Y., . A novel photon-enzyme cascade catalysis system based on hybrid HRP-CN/Cu₃(PO₄)₂ nanoflowers for degradation of BPA in water. Chem. Eng. J.. 2022;427:131808
[CrossRef] [Google Scholar]
Wu Z.F., Wang Z., Zhang Y., Ma Y.L., He C.Y., Li H., Chen L., Huo Q.S., Wang L., Li Z.Q., . Amino acids-incorporated nanoflowers with an intrinsic peroxidase-like activity. Sci. Rep.. 2016;6:22412.
[CrossRef] [Google Scholar]
Xue P., Liu X., Gu Y., Zhang W., Ma L., Li R., . Laccase-mediator system assembling co-immobilized onto functionalized calcium alginate beads and its high-efficiency catalytic degradation for acridine. Colloid. Surf. B. 2020;196:111348
[CrossRef] [Google Scholar]
Yang H., Wei W., Liu S., . Monodispersed silica nanoparticles as carrier for co-immobilization of bi-enzyme and its application for glucose biosensing. Spectrochim. Acta A Mol. Biomol. Spectrosc.. 2014;125:183-188.
[CrossRef] [Google Scholar]
Yu Y., Fei X., Tian J., Xu L., Wang X., Wang Y., . Self-assembled enzyme-inorganic hybrid nanoflowers and their application to enzyme purification. Colloids Surf. B Biointerfaces. 2015;130:299-304.
[CrossRef] [Google Scholar]
Zhang Z., Du Y., Kuang G., Shen X., Jia X., Wang Z., Feng Y., Jia S., Liu F., Bilal M., Cui J., . Lipase-Ca²⁺ hybrid nanobiocatalysts through interfacial protein-inorganic self-assembly in deep-eutectic solvents (DES)/water two-phase system for biodiesel production. Renew. Energ.. 2022;197:110-124.
[CrossRef] [Google Scholar]
Zhang L., Shi J., Jiang Z., Jiang Y., Qiao S., Li J., Wang R., Meng R., Zhu Y., Zheng Y., . Bioinspired preparation of polydopamine microcapsule for multienzyme system construction. Green Chem.. 2011;13:300-306.
[CrossRef] [Google Scholar]
Zhao F., Wang Q., Dong J., Xian M., Yu J., Yin H., Chang Z., Mu X., Hou T., Wang J., . Enzyme-inorganic nanoflowers/alginate microbeads: an enzyme immobilization system and its potential application. Process Biochem.. 2017;57:87-94.
[CrossRef] [Google Scholar]
Zhong L., Jiao X., Hu H., Shen X., Zhao J., Feng Y., Li C., Du Y., Cui J., Jia S., . Activated magnetic lipase-inorganic hybrid nanoflowers: a highly active and recyclable nanobiocatalyst for biodiesel production. Renew. Energ.. 2021;171:825-832.
[CrossRef] [Google Scholar]
Zhu L., Gong L., Zhang Y., Wang R., Ge J., Liu Z., Zare R.N., . Rapid detection of phenol using a membrane containing laccase nanoflowers. Chem. Asian J.. 2013;8:2358-2360.
[CrossRef] [Google Scholar]
Zhu X., Huang J., Liu J., Zhang H., Jiang J., Yu R., . A dual enzyme-inorganic hybrid nanoflower incorporated microfluidic paper-based analytic devices (μPADs) biosensor for sensitive visualized detection of glucose. Nanoscale. 2017;9:5658-5663.
[CrossRef] [Google Scholar]

Appendix A

Supplementary material

Supplementary data to this article can be found online at https://doi.org/10.1016/j.arabjc.2023.104770.

Appendix A

Supplementary material

The following are the Supplementary data to this article:

Supplementary data 1

Download

Show Sections

[1] Abdelhameed R.M., Emam H.E., . Design of ZIF(Co & Zn)@wool composite for efficient removal of pharmaceutical intermediate from wastewater. J. Colloid Interface Sci.. 2019;552:494-505.
[CrossRef] [Google Scholar]

[2] Altinkaynak, C., ÖZdemİR, N., ÖÇSoy, İ., 2021. A rational synthesis of magnetic nanoparticles incorporated horseradish peroxidase nanoflower and its use for the removal of phenol through oxidative coupling reaction with great reusability. Mugla. J. Sci. Techn. 7(2), 59−66. https://doi.org/10.22531/muglajsci.982993.

[3] Altinkaynak C., Tavlasoglu S., Kalin R., Sadeghian N., Ozdemir H., Ocsoy I., Ozdemir N., . A hierarchical assembly of flower-like hybrid Turkish black radish peroxidase-Cu²⁺ nanobiocatalyst and its effective use in dye decolorization. Chemosphere. 2017;182:122-128.
[CrossRef] [Google Scholar]

[4] Arif M., Yasin G., Shakeel M., Fang X., Gao R., Ji S., Yan D., . Coupling of bifunctional comn-layered double hydroxide@graphitic C₃N₄ nanohybrids towards efficient photoelectrochemical overall water splitting. Chem. Asian J.. 2018;13:1045-1052.
[CrossRef] [Google Scholar]

[5] Ariza-Avidad M., Salinas-Castillo A., Capitan-Vallvey L.F., . A 3D μPAD based on a multi-enzyme organic-inorganic hybrid nanoflower reactor. Biosens. Bioelectron.. 2016;77:51-55.
[CrossRef] [Google Scholar]

[6] Chung M., Nguyen T.L., Tran T.Q.N., Yoon H.H., Kim I.T., Kim M.I., . Ultrarapid sonochemical synthesis of enzyme-incorporated copper nanoflowers and their application to mediatorless glucose biofuel cell. Appl. Surf. Sci.. 2018;429:203-209.
[CrossRef] [Google Scholar]

[7] Cui J., Feng Y., Jia S., . Silica encapsulated catalase@metal-organic framework composite: A highly stable and recyclable biocatalyst. Chem. Eng. J.. 2018;351:506-514.
[CrossRef] [Google Scholar]

[8] Cui J., Jia S., . Organic–inorganic hybrid nanoflowers: a novel host platform for immobilizing biomolecules. Coord. Chem. Rev.. 2017;352:249-263.
[CrossRef] [Google Scholar]

[9] Cui J., Zhao Y., Liu R., Zhong C., Jia S., . Surfactant-activated lipase hybrid nanoflowers with enhanced enzymatic performance. Sci. Rep.. 2016;6:27928.
[CrossRef] [Google Scholar]

[10] Cui J., Ren S., Lin T., Feng Y., Jia S., . Shielding effects of Fe³⁺-tannic acid nanocoatings for immobilized enzyme on magnetic Fe₃O₄@silica core shell nanosphere. Chem. Eng. J.. 2018;343:629-637.
[CrossRef] [Google Scholar]

[11] Du Y., Jia X., Zhong L., Jiao Y., Zhang Z., Wang Z., Feng Y., Bilal M., Cui J., Jia S., . Metal-organic frameworks with different dimensionalities: an ideal host platform for enzyme@MOF composites. Coord. Chem. Rev.. 2022;454:214327
[CrossRef] [Google Scholar]

[12] Fang X., Tang Y., Ma Y.-J., Xiao G., Li P., Yan D., . Ultralong-lived triplet excitons of room-temperature phosphorescent carbon dots located on g-C₃N₄ to boost photocatalysis. Sci. China Mater.. 2022;66:664-671.
[CrossRef] [Google Scholar]

[13] Feng Y., Hu H., Wang Z., Du Y., Zhong L., Zhang C., Jiang Y., Jia S., Cui J., . Three-dimensional ordered magnetic macroporous metal-organic frameworks for enzyme immobilization. J. Colloid. Interf. Sci.. 2021;590:436-445.
[CrossRef] [Google Scholar]

[14] Feng Y., Du Y., Kuang G., Zhong L., Hu H., Jia S., Cui J., . Hierarchical micro- and mesoporous ZIF-8 with core–shell superstructures using colloidal metal sulfates as soft templates for enzyme immobilization. J. Colloid. Interf. Sci.. 2022;610:709-718.
[CrossRef] [Google Scholar]

[15] Fu M., Xing J., Ge Z., . Preparation of laccase-loaded magnetic nanoflowers and their recycling for efficient degradation of bisphenol A. Sci. Total. Environ.. 2019;651:2857-2865.
[CrossRef] [Google Scholar]

[16] Ge J., Lei J., Zare R.N., . Protein-inorganic hybrid nanoflowers. Nat. Nanotechnol.. 2012;7:428-432.
[CrossRef] [Google Scholar]

[17] Gu Y., Yuan L., Li M., Wang X., Rao D., Bai X., Shi K., Xu H., Hou S., Yao H., . Co-immobilized bienzyme of horseradish peroxidase and glucose oxidase on dopamine-modified cellulose–chitosan composite beads as a high-efficiency biocatalyst for degradation of acridine. RSC Adv.. 2022;11:23006-23016.
[CrossRef] [Google Scholar]

[18] Gul O.T., Ocsoy I., . Co-Enzymes based nanoflowers incorporated-magnetic carbon nanotubes: a new generation nanocatalyst for superior removal of cationic and anionic dyes with great repeated use. Environ. Technol. Inno.. 2021;24:101992
[CrossRef] [Google Scholar]

[19] Han J., Luo P., Wang L., Li C., Mao Y., Wang Y., . Construction of magnetic nanoflower biocatalytic system with enhanced enzymatic performance by biomineralization and its application for bisphenol A removal. J. Hazard. Mater.. 2019;380:120901
[CrossRef] [Google Scholar]

[20] Han J., Luo P., Wang L., Wu J., Li C., Wang Y., . Construction of a multienzymatic cascade reaction system of coimmobilized hybrid nanoflowers for efficient conversion of starch into gluconic acid. ACS Appl. Mater. Inter.. 2020;12:15023-15033.
[CrossRef] [Google Scholar]

[21] Hiner A.N., Hernandez-Ruiz J., Rodriguez-Lopez J.N., Arnao M.B., Varon R., Garcia-Canovas F., Acosta M., . The inactivation of horseradish peroxidase isoenzyme A2 by hydrogen peroxide: an example of partial resistance due to the formation of a stable enzyme intermediate. J. Biol. Inorg. Chem.. 2001;6:504-516.
[CrossRef] [Google Scholar]

[22] Huang Y., Ran X., Lin Y., Ren J., Qu X., . Self-assembly of an organic-inorganic hybrid nanoflower as an efficient biomimetic catalyst for self-activated tandem reactions. Chem. Commun.. 2015;51:4386-4389.
[CrossRef] [Google Scholar]

[23] Huang Y., Zou J., Lin J., Yang H., Wang M., Li J., Cao W., Yuan B., Ma J., . ABTS as both activator and electron shuttle to activate persulfate for diclofenac degradation: formation and contributions of ABTS+, SO4−, and OH. Environ. Sci. Technol. 2022
[CrossRef] [Google Scholar]

[24] Jiang Y., Zhai J., Zhou L., He Y., Ma L., Gao J., . Enzyme@silica hybrid nanoflowers shielding in polydopamine layer for the improvement of enzyme stability. Biochem. Eng. J.. 2018;132:196-205.
[CrossRef] [Google Scholar]

[25] Kaiser J., Feng Y., Bollag J., . Microbial metabolism of pyridine, quinoline, acridine, and their derivatives under aerobic and anaerobic conditions. Microbiol. Rev.. 1996;60(3):483-498.
[CrossRef] [Google Scholar]

[26] Kiani M., Mojtabavi S., Jafari-Nodoushan H., Tabib S.R., Hassannejad N., Faramarzi M.A., . Fast anisotropic growth of the biomineralized zinc phosphate nanocrystals for a facile and instant construction of laccase@Zn₃(PO₄)₂ hybrid nanoflowers. Int. J. Biol. Macromol.. 2022;204:520-531.
[CrossRef] [Google Scholar]

[27] Lee H.R., Chung M., Kim M.I., Ha S.H., . Preparation of glutaraldehyde-treated lipase-inorganic hybrid nanoflowers and their catalytic performance as immobilized enzymes. Enzyme Microb. Technol.. 2017;105:24-29.
[CrossRef] [Google Scholar]

[28] Li Y., Fei X., Liang L., Tian J., Xu L., Wang X., Wang Y., . The influence of synthesis conditions on enzymatic activity of enzyme-inorganic hybrid nanoflowers. J. Mol. Catal. B: Enzym.. 2016;133:92-97.
[CrossRef] [Google Scholar]

[29] Li H., Hou J., Duan L., Ji C., Zhang Y., Chen V., . Graphene oxide-enzyme hybrid nanoflowers for efficient water soluble dye removal. J. Hazard. Mater.. 2017;338:93-101.
[CrossRef] [Google Scholar]

[30] Li M., Luo M., Li F., Wang W., Liu K., Liu Q., Wang Y., Lu Z., Wang D., . Biomimetic copper-based inorganic–protein nanoflower assembly constructed on the nanoscale fibrous membrane with enhanced stability and durability. J. Phys. Chem. C. 2016;120:17348-17356.
[CrossRef] [Google Scholar]

[31] Li K., Wang J., He Y., Abdulrazaq M.A., Yan Y., . Carbon nanotube-lipase hybrid nanoflowers with enhanced enzyme activity and enantioselectivity. J. Biotechnol.. 2018;281:87-98.
[CrossRef] [Google Scholar]

[32] Li Y., Xie G., Qiu J., Zhou D., Gou D., Tao Y., Li Y., Chen H., . A new biosensor based on the recognition of phages and the signal amplification of organic-inorganic hybrid nanoflowers for discriminating and quantitating live pathogenic bacteria in urine. Sensors Actuators B: Chem.. 2018;258:803-812.
[CrossRef] [Google Scholar]

[33] Li Z., Zhang Y., Su Y., Ouyang P., Ge J., Liu Z., . Spatial co-localization of multi-enzymes by inorganic nanocrystal-protein complexes. Chem. Commun.. 2014;50:12465-12468.
[CrossRef] [Google Scholar]

[34] Li P., Zhang T., Mushtaq M.A., Wu S., Xiang X., Yan D., . Research progress in organic synthesis by means of photoelectrocatalysis. Chem. Rec.. 2021;21:841-857.
[CrossRef] [Google Scholar]

[35] Li C., Zhao J., Zhang Z., Jiang Y., Bilal M., Jiang Y., Jia S., Cui J., . Self-assembly of activated lipase hybrid nanoflowers with superior activity and enhanced stability. Biochem. Eng. J.. 2020;158:107582
[CrossRef] [Google Scholar]

[36] Liu X., Xue P., Jia F., Shi K., Gu Y., Ma L., Li R., . A novel approach to efficient degradation of indole using co-immobilized horseradish peroxidase-syringaldehyde as biocatalyst. Chemosphere. 2021;262:128411
[CrossRef] [Google Scholar]

[37] Ma, N., Xue, P., Li R., Ma L., Shi K., . Flower-like hybrid composite MoS₂/NH₂-MIL-101(Cr): a highly efficient photocatalyst for degrading indole under visible light. J. Mater. Res.. 2021;36:4460-4474.
[CrossRef] [Google Scholar]

[38] Mohammad M., Ahmadpoor F., Shojaosadati S.A., . Mussel-inspired magnetic nanoflowers as an effective nanozyme and antimicrobial agent for biosensing and catalytic reduction of organic dyes. ACS Omega. 2020;5:18766-18777.
[CrossRef] [Google Scholar]

[39] Mushtaq M.A., Kumar A., Yasin G., Arif M., Tabish M., Ibraheem S., Cai X., Ye W., Fang X., Saad A., Zhao J., Ji S., Yan D., . 3D interconnected porous Mo-doped WO₃@CdS hierarchical hollow heterostructures for efficient photoelectrochemical nitrogen reduction to ammonia. Appl. Catal. B-Environ.. 2022;317:121711
[CrossRef] [Google Scholar]

[40] Ocsoy I., Dogru E., Usta S., . A new generation of flowerlike horseradish peroxides as a nanobiocatalyst for superior enzymatic activity. Enzyme. Microb. Technol.. 2015;75–76:25-29.
[CrossRef] [Google Scholar]

[41] Pitzalis F., Monduzzi M., Salis A., . A bienzymatic biocatalyst constituted by glucose oxidase and Horseradish peroxidase immobilized on ordered mesoporous silica. Micropor. Mesopor. Mater.. 2017;241:145-154.
[CrossRef] [Google Scholar]

[42] Razzaghi M., Homaei A., Vianello F., Azad T., Sharma T., Nadda A.K., Stevanato R., Bilal M., Iqbal H.M.N., . Industrial applications of immobilized nano-biocatalysts. Bioprocess. Biosyst. Eng.. 2022;45:237-256.
[CrossRef] [Google Scholar]

[43] Ren W., Fei X., Tian J., Li Y., Jing M., Fang H., Xu L., Wang Y., . Multiscale immobilized lipase for rapid separation and continuous catalysis. New J. Chem.. 2018;42:13471-13478.
[CrossRef] [Google Scholar]

[44] Ren W., Li Y., Wang J., Li L., Xu L., Wu Y., Wang Y., Fei X., Tian J., . Synthesis of magnetic nanoflower immobilized lipase and its continuous catalytic application. New J. Chem.. 2019;43:11082-11090.
[CrossRef] [Google Scholar]

[45] Rong J., Zhang T., Qiu F., Zhu Y., . Preparation of efficient, stable, and reusable laccase–Cu₃(PO₄)₂ hybrid microspheres based on copper foil for decoloration of congo red. ACS Sustain. Chem. Eng.. 2017;5:4468-4477.
[CrossRef] [Google Scholar]

[46] Sharma N., Parhizkar M., Cong W., Mateti S., Kirkland M.A., Puri M., Sutti A., . Metal ion type significantly affects the morphology but not the activity of lipase–metal–phosphate nanoflowers. RSC Adv.. 2017;7:25437-25443.
[CrossRef] [Google Scholar]

[47] Sun H., Huang W., Yang H., . Co-immobilization of laccase and mediator through a self-initiated one-pot process for enhanced conversion of malachite green. J. Colloid Interf. Sci.. 2016;471:20-28.
[CrossRef] [Google Scholar]

[48] Sun K., Huang Q., Li S., . Transformation and toxicity evaluation of tetracycline in humic acid solution by laccase coupled with 1-hydroxybenzotriazole. J. Hazard. Mater.. 2017;331:182-188.
[CrossRef] [Google Scholar]

[49] Wang C., Hu B., Wang J., Gao J., Li G., Xiong W.W., Zou B., Suzuki M., Aratani N., Yamada H., Huo F., Lee P.S., Zhang Q., . Rewritable multilevel memory performance of a tetraazatetracene donor-acceptor derivative with good endurance. Chem. Asian J.. 2015;10:116-119.
[CrossRef] [Google Scholar]

[50] Wen H., Zhang L., Du Y., Wang Z., Jiang Y., Bian H., Cui J., Jia S., . Bimetal based inorganic-carbonic anhydrase hybrid hydrogel membrane for CO₂ capture. J. CO₂ Util.. 2020;39:101171
[CrossRef] [Google Scholar]

[51] Wu J., Ma X., Li C., Zhou X., Han J., Wang L., Dong H., Wang Y., . A novel photon-enzyme cascade catalysis system based on hybrid HRP-CN/Cu₃(PO₄)₂ nanoflowers for degradation of BPA in water. Chem. Eng. J.. 2022;427:131808
[CrossRef] [Google Scholar]

[52] Wu Z.F., Wang Z., Zhang Y., Ma Y.L., He C.Y., Li H., Chen L., Huo Q.S., Wang L., Li Z.Q., . Amino acids-incorporated nanoflowers with an intrinsic peroxidase-like activity. Sci. Rep.. 2016;6:22412.
[CrossRef] [Google Scholar]

[53] Xue P., Liu X., Gu Y., Zhang W., Ma L., Li R., . Laccase-mediator system assembling co-immobilized onto functionalized calcium alginate beads and its high-efficiency catalytic degradation for acridine. Colloid. Surf. B. 2020;196:111348
[CrossRef] [Google Scholar]

[54] Yang H., Wei W., Liu S., . Monodispersed silica nanoparticles as carrier for co-immobilization of bi-enzyme and its application for glucose biosensing. Spectrochim. Acta A Mol. Biomol. Spectrosc.. 2014;125:183-188.
[CrossRef] [Google Scholar]

[55] Yu Y., Fei X., Tian J., Xu L., Wang X., Wang Y., . Self-assembled enzyme-inorganic hybrid nanoflowers and their application to enzyme purification. Colloids Surf. B Biointerfaces. 2015;130:299-304.
[CrossRef] [Google Scholar]

[56] Zhang Z., Du Y., Kuang G., Shen X., Jia X., Wang Z., Feng Y., Jia S., Liu F., Bilal M., Cui J., . Lipase-Ca²⁺ hybrid nanobiocatalysts through interfacial protein-inorganic self-assembly in deep-eutectic solvents (DES)/water two-phase system for biodiesel production. Renew. Energ.. 2022;197:110-124.
[CrossRef] [Google Scholar]

[57] Zhang L., Shi J., Jiang Z., Jiang Y., Qiao S., Li J., Wang R., Meng R., Zhu Y., Zheng Y., . Bioinspired preparation of polydopamine microcapsule for multienzyme system construction. Green Chem.. 2011;13:300-306.
[CrossRef] [Google Scholar]

[58] Zhao F., Wang Q., Dong J., Xian M., Yu J., Yin H., Chang Z., Mu X., Hou T., Wang J., . Enzyme-inorganic nanoflowers/alginate microbeads: an enzyme immobilization system and its potential application. Process Biochem.. 2017;57:87-94.
[CrossRef] [Google Scholar]

[59] Zhong L., Jiao X., Hu H., Shen X., Zhao J., Feng Y., Li C., Du Y., Cui J., Jia S., . Activated magnetic lipase-inorganic hybrid nanoflowers: a highly active and recyclable nanobiocatalyst for biodiesel production. Renew. Energ.. 2021;171:825-832.
[CrossRef] [Google Scholar]

[60] Zhu L., Gong L., Zhang Y., Wang R., Ge J., Liu Z., Zare R.N., . Rapid detection of phenol using a membrane containing laccase nanoflowers. Chem. Asian J.. 2013;8:2358-2360.
[CrossRef] [Google Scholar]

[61] Zhu X., Huang J., Liu J., Zhang H., Jiang J., Yu R., . A dual enzyme-inorganic hybrid nanoflower incorporated microfluidic paper-based analytic devices (μPADs) biosensor for sensitive visualized detection of glucose. Nanoscale. 2017;9:5658-5663.
[CrossRef] [Google Scholar]

Construction of novel bienzyme-inorganic hybrid nanoflowers beads and their application in the efficient degradation of acridine

Abstract

Keywords

Biocatalysis

Cascade reaction

Acridine degradation

Wastewater treatment

Bienzyme-inorganic hybrid nanoflowers beads;

1 Introduction

2 Materials and methods

2.1 Chemicals and enzyme

2.2 Preparation of biE-HNFs-beads

2.2.1 Preparation of Ce-Cs@DA beads

2.2.2 Adsorption of copper ions on the surface of Ce-Cs@DA beads.

2.2.3 Preparation of biE-HNFs-beads

2.3 The catalytic activity and encapsulation yield of biE-HNFs-beads

2.4 Evaluation of the kinetic parameters of the biE-HNFs-beads

2.5 Degradation of acridine by biE-HNFs-beads

2.6 The properties of biE-HNFs-beads

2.6.1 Thermal and pH stability of biE-HNFs-beads

2.6.2 Storage stability of biE-HNFs-beads

2.6.3 Reusability of biE-HNFs-beads

2.7 Characterization of biE-HNFs-beads

3 Results and discussion

3.1 Synthesis mechanism of biE-HNFs-beads

3.2 Characterisation of biE-HNFs-beads

3.3 The kinetics of biE-HNFs-beads

3.4 The optimization of formation conditions for biE-HNFs-beads

3.4.1 The effect of synthesis temperature

3.4.2 The effect of the concentration ratios of HRP:GOD in solution

3.4.3 The effect of bienzyme concentration

3.4.4 The effect of different preparation methods

3.5 Properties of biE-HNFs-beads

3.5.1 Effect of reaction parameters on the catalytic degradation of acridine

3.5.2 Stability study of biE-HNFs-beads

3.6 Possible degradation products and pathways of acridine

4 Conclusions

CRediT authorship contribution statement

Acknowledgments

Declaration of Competing Interest

References

Appendix A

Supplementary material

Appendix A

Supplementary material

Supplementary data 1

Suggested read for related articles: