1. Introduction
Urbanization, growing population and industrialization have led to a considerable release of manmade pollutants that due to their synthetic origin are recalcitrant to biodegradation, causing their persistence and accumulation in the environment. In addition, most of them are highly toxic posing wildlife and human health at risk. Therefore, the removal of the aforementioned pollutants from wastewater before being discharged in the environment is an urgent need. However, conventional wastewater treatments are inefficient in the removal of such type of pollutants and emerging technologies are either costly, non-environmentally friendly or non-feasible on a large scale. This has driven forward the search for new efficient, cost-effective and ecological wastewater treatment technologies. In this sense, the use of ligninolytic enzymes produced by certain microorganisms are particularly interesting due their broad substrate specificity and natural origin. Among the different existing ligninolytic enzymes, laccases (E.C. 1.10.3.2; p-bezenediol: dioxygen oxidoreductases) have attracted increasing interest during the last decades on account of they only need environmental molecular oxygen to exert their catalytic action producing water as the only by-product. Their catalytic site consists of four copper atoms that are classified, according to their electron paramagnetic resonance and UV-vis spectroscopy characteristics, in three types and located at different sites.[1] The type 1 copper (T1) is involved in the oxidation of the substrate. The type 2 copper (T2) and the two type 3 coppers (T3) form a trinuclear cluster where the molecular oxygen is reduced to water. The connection between the two sites (i.e., the T1 site and the T2-T3 cluster site) is guaranteed by a tripeptide coupling of amino acids (His-Cys-His) (Figure 1).[1]
Laccases are widely distributed in Nature and, thus, they have been found in bacteria, fungi, higher plants and insects.[2] Among them, laccases from white-rot fungi are particularly interesting since they have the highest redox potential. In addition, fungal laccases are often glycosylated what gives them conformational stability and protection from inactivation by radicals and proteolysis.[3,4] Also, recently the mechanism of molecular oxygen reduction for high-redox potential laccases has been described and has been found that contrary to the low-redox potential laccases, the T1 copper reduction is very fast[5] and, hence, their high appeal for biotechnological processes. Consequently, laccase production and application as a biocatalyst have been increasingly reported in the literature.[4,6,7] However, despite laccase production cost is an important issue for its industrial exploitation[8], techno-economic analyses have been scarcely reported
On the other hand, regarding laccase applications, the use of free enzymes for wastewater treatment holds different drawbacks such as non-reusability, high cost, lack of long-term stability and sensibility to different denaturing agents (e.g., pH, temperature, mechanical stress and inhibiting compounds that may occur in wastewater). In fact, there is no recorded industrial application of free laccase.[9] The above-mentioned drawbacks can be solved by immobilising laccase enzymes in/on solid carriers. The selection of a suitable solid carrier for laccase immobilisation is essential for the efficiency of the developed bioprocess. An ideal carrier should protect both laccase structure and activity under different operational and environmental conditions while keeping its own physical integrity. In addition, it should have high affinity for laccase enzyme, be inert, easily available, low-cost and environmentally friendly. Recently, Kyomuhimbo and Brink (2023) have reviewed the different supports used for laccase immobilisation.[10]
Different immobilisation techniques than can be categorised in physical (adsorption, encapsulation, entrapment) and chemical (covalent binding, crosslinking) and different solid carriers have been tested to make laccase enzymes reusable and increase their stability as recently reviewed by Alvarado-Ramírez et al. (2021).[11]Also, Zhou et al. (2021) reviewed the different immobilisation methods and carriers used to immobilise laccase as well as the application of the immobilised laccases in water purification.[12] Each immobilisation protocol presents its advantages and disadvantages (Table 1). So, there is not a preferred method for laccase immobilisation and will depend on the laccase source, the carrier, the intended application and the operating conditions.[13] Therefore, the design of new laccase immobilisation protocols is still worth of investigation. Current trends are oriented towards the use of a low quantity of laccase enzyme, minimum chemical reagents and biodegradable and low-cost materials, such as agro-industrial wastes, as solid carriers.[9] To scale up the wastewater treatment process, the immobilised laccases are placed in different reactor configurations. This review explores the reported use of immobilised-laccase reactors for pollutant removal and wastewater treatment from 2020 up to date.