Efficient removal of various textile dyes from wastewater by novel thermo-halotolerant laccase
Graphical abstract
Introduction
Synthetic dyes are a critical part of various industries, such as textile, paper, plastic, printing, and leather that possess severe toxic effects on the environment. These hazardous chemicals are highly persistent, which lead to water pollution, change the ecosystem balance and have a severe impact on the animal, plant, and human health (Ariaeenejad et al., 2021, Rahimi et al., 2020). Azo dyes are considered as the largest class of synthetic dyes used in textile manufacturing which around 10–15% of them are lost through dyeing process (Sarkar et al., 2017). Triphenylmethane and anthraquinone dyes are other groups of synthetic dyes that cause severe environmental pollution problems by releasing large amounts of potentially toxic compounds (Navas et al., 2020). Removing hazardous contaminants before discharge to the environment is a challenging problem and a recent global priority. Therefore, developing a low-cost and eco-friendly way of dealing with textile dyeing waste has gained popularity (Ariaeenejad et al., 2021). In this respect, laccase enzymes have attracted increasing attention for dye detoxification and degradation (Chmelová and Ondrejovič, 2016, Du et al., 2020, Iark et al., 2019, Mehandia et al., 2020, Si et al., 2013, Yang et al., 2020).
Laccases (benezenediol: oxygen oxidoreductase, EC 1.10.3.2) belong to the family of multicopper oxidases that broadly spread in bacteria, plants, and fungi (Mate and Alcalde, 2017). These enzymes are best known for their ability to act on a broad range of substrates and degrade persistent environmental pollutants. Laccases can oxidize a variety of phenolic and non-phenolic compounds by reducing the molecular oxygen to water (Guan et al., 2018). They are beneficial enzymes employed in multipurpose applications from bio-cells and bio-sensors to bioremediation, dye decolorization, food, and paper industries (Mate and Alcalde, 2017). There have been many reports of laccase for effective biodegradation and detoxification of synthetic dyes up to now (Du et al., 2020, Song et al., 2017, Zhang et al., 2012). The wastewater from the dying process usually contains high concentrations of metal ions, salts, organic solvents and involves high temperatures and neutral or alkaline pHs, limiting the activity of most laccases (Santhanam et al., 2011). Thus, looking for robust laccases that can tolerate these extreme conditions is an essential issue for industrial dye decolorization.
Several approaches have been applied to identify the novel laccases for various industrial purposes, especially dye decolorization. However, a traditional method for producing novel laccases is using culturable microorganisms that include microorganism cultivation, production, purification, and studying the characteristics of the enzyme (Liu et al., 2019). Due to the poor cultivability of the vast number of microorganisms and the high cost of this approach, it is not desirable to produce laccases on a large scale (Liu et al., 2019). Therefore, metagenomics have proven to overcome the limitations of culture-dependent methods and led to identifying novel laccases capable of degrading pollutant and used for bioremediation (Ufarté et al., 2015). Up until now, various laccases have been successfully mined from soil, marine, and plant sludge metagenome (Ausec et al., 2017, Zhong et al., 2013). Based on the rapid developments in advanced sequencing techniques, the multi-stage screening pipeline coupled with the bioinformatic tools showed the strong power for the discovery of diverse and novel improved enzymes from the considerable diversity of unculturable microbes for a wide range of applications such as biorefinery, lignocellulose-based and, food industries in the recent years (Ariaeenejad et al., 2020b, Ariaeenejad et al., 2020c, Motahar et al., 2020, Motamedi et al., 2021)
This study aimed to introduce a novel thermostable laccase from tannery wastewater metagenomic data along with its potent application in decolorization of various dyes from effluents. The enzyme was first characterized to find its optimum temperature and pH, then its storage stability under optimum working conditions was evaluated. This novel laccase enzyme showed high resistance to different concentrated metal ions, organic solvents, and common inhibitors. Furthermore, the application of PersiLac2 for removing three major classes of dyes (azo, tri-phenylmethane, and anthraquinone) and its detoxification effect on germination of the rice grain in the presence of these three dye groups were confirmed.
Section snippets
Dyes and chemicals
The various dyes used in this study were from Merck Co. (Table 1). The mediators including ABTS (2,2́-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)), HBT (hydroxybenzotriazole), and Syringaldazine were Sigma-Aldrich products. The metal ions, dithiothreitol (DTT), sodium dodecyl sulfate (SDS), sodium azide (NaN3), ethylenediaminetetraacetic acid (EDTA), acetone, acetonitrile, iso-propanol, ethanol, ethylene glycol, iso-butanol, methanol, glycerol and dimethyl sulfoxide (DMSO) were purchased
Screening and identification of novel PersiLac2
It was expected that the bacterial metagenome of the tannery wastewater encoded for the novel and special laccases with the high activity in harsh conditions. By employing the MeTarEnz, CDD, and Phyre2 tools, the final laccase candidate was selected and named PersiLac2.
Analysis of the predicted enzyme from metagenome versus conserved domains using the PSSM models in the CDD, revealed its high similarity with laccase superfamily members (Ariaeenejad et al., 2020a). By 3D modeling of PersiLac2
Conclusion
This study introduced a novel thermostable and halo-tolerant laccase enzyme that showed resistance against metal ions, surfactants and could effectively and practically be utilized for dye removal from wastewater. The enzyme was highly active and stable under various harsh conditions and was also able to decolorize a high concentration of azo, anthraquinone, and triphenylmethane dyes in a short time without need to addition of redox mediators. Moreover, effective decolorization of the real
CRediT authorship contribution statement
Elaheh Motamedi: Methodology, Investigation, Writing - original draft. Kaveh Kavousi: Writing - review & editing. Seyedeh Fatemeh Sadeghian Motahar: Investigation, Writing - original draft. Mohammad Reza Ghaffari: Formal analysis, Software, Data curation. Atefeh Sheykh Abdollahzadeh Mamaghani: Investigation, Methodology, Writing - original draft. Ghasem Hosseini Salekdeh: Resources. Shohreh Ariaeenejad: Conceptualization, Methodology, Supervision, Investigation, Writing - original draft.
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.
Acknowledgement
This research was supported by a grant from Agricultural Biotechnology Research Institute of Iran (ABRII) and Iran National Science Foundation (INSF) (funding reference number 99016942). The authors gratefully acknowledge the help of HARIR SEMNAN DYEING & PRINTING CO., especially Mr. A. Sarsharzadeh, and Mr. A. A. Khakbaz from the administrative board.
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