Agro-industrial-Produced Laccase for Degradation of Diclofenac and Identification of Transformation Products

Abstract

A widely used anti-inflammatory drug, diclofenac (DCF), is recalcitrant in many environmental compartments and poses threat to several aquatic and terrestrial organisms. Enzymatic degradation of emerging contaminants which are often micropollutants, has gained interest for the past few years. However, production of enzymes often incurs high costs. In this study, ligninolytic enzyme laccase was produced by white rot fungi Tremetes versicolor (ATCC 20869) using agro-industrial residues, apple pomace (AP), pulp and paper solid waste (PPSW), and alfa fibers as substrates. Various known inducers for laccase production, such as tween 80 (0.1% (w/w)), veratryl alcohol (3 mM Kg–1), CuSO4 (3 mM Kg–1), and phenol red (3 mM Kg–1) were used to enhance laccase production. A maximum laccase activity of 49.16 ± 4.5, 52.4 ± 2.2, and 14.26 ± 0.8 U/gds (units/gram dry substrate) was obtained from apple pomace, PPSW, and alfa plant fibers, respectively, at optimal experimental conditions. Further, the kinetics of the laccase mediated degradation of DCF was studied. At environmentally relevant concentration of DCF (500 μg L–1), laccase-catalyzed degradation followed first-order kinetics. At environmentally relevant concentrations pH of 4.5 and temperature of 50 °C was found to be optimal for the effective degradation of DCF with laccase. 3′-Hydroxydiclofenac, 4′-hydroxydiclofenac, and 5-hydroxydiclofenac were identified as the major transformation products during the initial 5 h of degradation. However, after 24 h of degradation, neither DCF nor any transformation products were identified so that the proposed degradation mechanism involved hydroxylation followed by ring opening and final mineralization to CO2, NH3, and H2O and HCl.

Fungal Laccase Production from Lignocellulosic Agricultural Wastes by Solid-State Fermentation: A Review

“…Aromatic inducers stimulated response signal recognition in white rot fungi, resulting in an intense biological response that induced secondary metabolism, leading to increased laccase concentration [77]. Other compounds, such as Tween 80 and veratryl alcohol, were used to enhance laccase production [34]. Xylidine was reported to increase laccase production more efficiently than copper [3].…”

“…The well-studied inducers of increased laccase production are summarized in Table 3. Table 3 shows that copper is an excellent inducer for laccase production when dosed as CuSO 4 to increase laccase production [78] because laccase is blue copper oxidase, containing four copper atoms per molecule, and the addition of copper may lead to the activation of the metal, resulting in the expression of laccase genes [34]. Unfortunately, most inducers known to date have disadvantages such as toxic effects, high production costs, and environmental pollution.…”

“…However, problems such as low-volume production and high cost remain unsolved, which hinder the wide application of laccase [48]. To solve these problems, researchers studied laccase production by SSF using lignocellulosic agricultural waste as a substrate (Table 1) because SSF is more compatible with the natural growth of the strain and the lignocellulosic substrates are inexpensive, readily available, and environmentally friendly [10,34]. However, it is well-known that scaling up of SSF still has some difficulties, especially for SSF bioreactors.…”

“…One review highlighted the potential of lignocellulosic materials to be used in different applications involving biofuels, enzymes, chemicals, pulp and paper, animal feed, and composites [31]. A variety of lignocellulosic residues associated with agriculture (e.g., sunflower seed hulls, sugarcane bagasse, sawdust waste, apple pomace, cotton stalks) have been studied in this regard [20,[32][33][34][35][36]. There have been more than 20 review papers published since 2010, and different topics about laccase have been summarized and discussed, including characteristics, expression and regulation, molecular design, production, and applications.…”

TiO2-reduced graphene oxide nanocomposites for the trace removal of diclofenac

“…DCF, [2-(2,6-dichlorophenylamino) phenylacetic acid] is one of the popular NSAIDs in medical treatment as an analgesic, antiarthritic and antirheumatic agent apart from its veterinary use. The annual human consumption of DCF has been estimated globally to be ⁓ 1500 tons [2]. It has been shown that about 15-30% of the consumed DCF is excreted from the human body unmodified and enters the aquatic environment [3].…”