A novel approach to efficient degradation of indole using co-immobilized horseradish peroxidase-syringaldehyde as biocatalyst
Graphical abstract
Introduction
Indole, a typical nitrogen heterocyclic compound, is often the existence of pharmaceutical, coking and dying industrial wastewater. As refractory organic in water, it is difficult to be degraded by microorganisms in natural conditions owing to its stable chemical structure (van Loosdrecht and Brdjanovic, 2014; Ben Hammouda et al., 2016; Chen et al., 2013). Once discharged into the natural environment, the indole in wastewater could cause irreversible damage to cells by accumulating in organisms, resulting in the adverse reaction of teratogenic, carcinogenic and mutagenic on animals and humans (Huang et al., 2016). Therefore, it is necessary to develop an efficient, green and economical technique of degrading indole to avoid its harm to human health and ecological environment. Nowadays, the conventional removal methods for indole including physical, chemical as well as biological methods have been experimentally studied, such as adsorption, chemical oxidation, electrocatalytic oxidation and enzymatic degradation (Ning et al., 2017; Huang and Wang, 2007; Liu et al., 2018; Ma et al., 2019). It is commonly recognized that physical and chemical treatments have many inadequacies of high running costs, complex operating conditions and serious secondary pollution and so on. By contrast, the enzymatic degradation has superior advantages of mild conditions, low energy consumption and environmental friendliness (Yi et al., 2019). In recent years, the removal of aromatic compounds from wastewater catalyzed by oxidoreductases has been investigated (Salami et al., 2018; Zheng et al., 2016; Farias et al., 2017a) and the degradation of several nitrogen heterocyclic compounds has been discussed (Bayramoglu et al., 2012, 2019).
Horseradish peroxidase has been widely used in biocatalytic degradation for phenolic and aniline compounds in the presence of hydrogen peroxide and has displayed great potential in water treatment (Xu et al., 2013; Niu et al., 2013; Bodalo et al., 2006). Up to now, it has been rarely explored in the degradation of aromatic and nitrogen heterocyclic compounds due to its low oxidation-reduction potential (Ji et al., 2016). It was found that some small organic molecules named redox mediator with low redox potential such as 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 1-hydroxybenzotriazole (HBT) and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) are capable of broadening the substrate range and improving the catalytic performance of redox enzymes (Zeng et al., 2017; Melo and Dezotti, 2013; Saladino et al., 2013). The redox mediators participated in the catalytic reactions and improved the catalytic performance of enzymes by promoting electrons or hydrogen atoms transfer in the redox process (Canas and Camarero, 2010). For example, Chen et al. (2014) investigated the biocatalytic oxidation of polycyclic aromatic hydrocarbons (PAHs) in water using HRP as catalyst, and it showed that the transformation for anthracene of 1 mg/mL was 48.7% without mediator HBT, while the transformation reached 80.9% in the presence of 1 mM HBT. The results demonstrated that organic molecule HBT as redox mediator possessed synergistic catalytic effect on the catalysis of HRP, which can enhance the enzyme-catalyzed reaction rate. It can be predicted that the deep and extensive research on enzyme-mediator system will strongly promote the application of biocatalysis technology in wastewater treatment.
The enzyme’s immobilization is necessary to realize the practice application of its biocatalysis because the free enzyme is itself unstable and difficult to recover and reuse, where the support material and immobilization way are very critical to the catalytic performance of the immobilized enzyme (Sheldon and van Pelt, 2013). At present, the solid materials of alginate, magnetic nanoparticles, hybrid materials, electrospun microfibrous membranes were applied to remove drugs, phenols and dyes in wastewater (Ratanapongleka and Punbut, 2017; Shi et al., 2014; Xu et al., 2013; Farias et al., 2017b; Arica et al., 2019). For example, Wang et al. (2015) immobilized HRP on the ethylene diamine and chitosan polyacrylonitrile modified by glutaraldehyde crosslinking to remove 2,4-dichlorophenol in water. It was showed that the degradation rate was up to 90% above by using the immobilized HRP to treat 3 mM 2,4-dichlorophenol. The HRP was immobilized on NH2-modified magnetic Fe3O4/SiO2 particles using glutaraldehyde as a crosslinking agent (Chang and Tang, 2014). Its catalytic activity was held, and the removal of 80% was achieved in 180 min for treating 0.2 mM 2,4-dichlorophenol in the presence of hydrogen peroxide at 30 °C and pH 6.4. Bayramoglu and Arica (2008) investigated the immobilization of HRP on magnetic poly (glycidylmethacrylate-co-methylmethacrylate) beads via glutaraldehyde coupling to remove phenolic compounds from aqueous solution in the presence of hydrogen peroxide. The immobilized HRP was more resistant to temperature inactivation than that of free HRP. In the catalytic application of enzyme-mediator system, the co-immobilization of both on a support is of great significance. If free enzyme and free mediator were used with a direct mix, neither of both could be separated from the reaction system for recycling, which could result in high use cost, extremely complicated and difficult operation as well as the risk of environmental pollution from poisonous mediators. Liu et al. (2015) immobilized mediator ABTS on silica nanoparticles, and the results showed that the decolorization yield of indigo carmine in the immobilized ABTS-free laccase system was almost the same as that of free ABTS-free laccase system. The metal organic framework material was employed to immobilize ABTS for laccase-catalyzed decolorization of indigo carmine (Liu et al., 2017), and the decolorization yields of 95% and 94% were obtained by laccase in combination with free ABTS or immobilized ABTS, respectively. The mediator ABTS was cross-linked via electron irradiation in a frozen aqueous polyacrylate mixture (Jahangiri et al., 2014). It showed that the immobilized ABTS remained functional in accelerating the degradation of bisphenol A by laccase. It can be seen that the immobilized ABTS still possessed its synergistic catalytic function of improving laccase catalytic activity. At present, most studies only focus on the immobilization of a single enzyme or a single redox mediator, and there are few reports about the co-immobilization of enzyme and mediator on the appropriate support. Gao et al. (2018) employed amino-functionalized Fe3O4 nanoparticles for co-immobilization of laccase and TEMPO by glutaraldehyde cross-linking. The results showed that the maximum decolorization rate reached 77.41% using the co-immobilized laccase-TEMPO nanoparticles to decolorize acid fuchsin, and the co-immobilized laccase-TEMPO nanoparticles could be reused retaining above 50% residual activity after eight cycles of operation. However, the co-immobilization for the enzyme and mediator by cross-linking could result in the direct contact between laccase and TEMPO, which might give rise to the active loss of laccase from the consecutive oxidation reaction between laccase and TEMPO. We immobilized laccase and ABTS into the dual-functionalized cellulose beads to degrade indole in water (Gu et al., 2019). The results showed that the co-immobilized laccase-ABTS possessed considerable operational stability for reuse, which has realized the synchronous recovery of the enzyme and ABTS and avoided the potential secondary contamination from ABTS. Nevertheless, the studies on co-immobilization of HRP and mediator syringaldehyde have never been reported yet.
Sodium alginate of a linear anionic copolymer can form an “egg-box” shape structure by crosslinking with divalent cations of Ca2+, Cu2+ and Ba2+ and so on (Lee and Mooney, 2012). It was usually employed for the immobilization of enzymes due to its biocompatibility, low toxicity and low cost (Daâssi et al., 2014; Noreen et al., 2015). In this study, the functionalized calcium alginate composite beads grafted with glycidyl methacrylate and dopamine were employed for the co-immobilization of HRP and redox mediator syringaldehyde (SY) as a novel biocatalyst to catalytically degrade indole in water. It was found for the first time that indole can be effectively degraded by HRP-SY system, while it can hardly be degraded by HRP alone. Different from synthetic organic mediators of ABTS, HBT and TEMPO, SY as a natural redox mediator possessed the advantages of easy source, low cost, non-toxic and environmental friendliness. Therefore, the co-immobilized HRP-SY system was an excellent biocatalyst for the degradation of indole in wastewater. The reusability of the biocatalyst and factors affecting the degradation rate of indole were studied in detail.
Section snippets
Enzyme and chemicals
HRP (>200 U/mg) from Wasabi was obtained by Tci. SY and dopamine hydrochloride were purchased from Ark Pharm. Sodium alginate and glycidyl methacrylate (GMA) were supplied by Alfa Aesar and Adamas Reagent, respectively. Ammonium peroxy disulfate (APS), N-hydroxysuccinimide (NHS), hydrogen peroxide solution (H2O2) and calcium chloride dehydrate (CaCl2·2H2O) were purchased from Sinopharm Chemical Reagent Co., Ltd. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and indole (99%)
Characteristics
The process diagram of encapsulating redox mediator SY into the functionalized calcium alginate composite beads was shown in Fig. 1A. Sodium alginate was functionalized with GMA by emulsion polymerization using APS as an initiator to graft active epoxy groups. The epoxy groups can couple with the groups of sulfhydryl, amino, carboxyl and hydroxyl of enzyme molecules (Akkaya and Uslan, 2010) to achieve the covalent immobilization for HRP. Subsequently, the dopamine containing catechol groups was
Conclusions
It was firstly found that SY molecule possessed significant synergistic catalytic action as a redox mediator for indole degradation by HRP, resulting in the effective degradation of indole in water by HRP-SY system in the existence of H2O2. The HRP/PDGC-SY beads have been prepared by the co-immobilization of HRP and SY on the functionalized calcium alginate composite beads grafted with GMA and dopamine and used as biocatalyst for degrading indole. The HRP/PDGC-SY beads have displayed excellent
Credit author statement
Xueping Liu: Methodology, Investigation, Data analysis, Writing-original draft. Ping Xue: Conceptualization, Methodology, Writing-review & editing. Feng Jia: Methodology, Investigation. Keren Shi: Guidance on the characterization, Investigation. Yaohua Gu: Investigation. Lan Ma: Guidance on the characterization. Rui Li: Guidance on the characterization.
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.
Acknowledgments
This work was financially supported by the National Natural Science Foundation of China (No.21961028) and the Science and Technology Support Project of Ningxia Province (NX015076).
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