Nowadays, the economic scenario is mostly based on fossil resources with a continuous increase of both their demand and their relative market price with a negative impact on the environment. The Green Transition is pushing towards the use of renewable resources to produce heat and energy, for a better employment of natural resources, such as solar, wind and hydropower energy. To produce chemicals, biomasses are used as sustainable alternatives to fossil resources by biorefining, which is one of the main drivers for the bio-based economy (Ciliberti et al. 2020; Dubois and Gomez San Juan 2016). The European Directive 2009/23/EC has defined biomass as “the biodegradable fraction of products, waste and residues from biological origin from agriculture (including plant and animal substances), forestry and related industries including fisheries and aquaculture, as well as the biodegradable fraction of industrial and municipal waste”(EUR-Lex 2009). The use of agro-industrial waste has been reported both in submerged and solid-state fermentation (SSF) due to the accessibility of insoluble biomass (Adekunle et al. 2016). SSF has many advantages, such as higher product titer, lower wastewater output, simpler fermentation media, reduced energy requirements (Lizardi-Jiménez and Hernández-Martínez 2017).
Industrially relevant enzymes can be produced by solid-state fermentation for their deployment in organic synthesis. In particular, the selective oxidation of alcohols to the corresponding carbonyl compounds is of utmost importance in both academia and industry. Primary alcohols are directly oxidized to carboxyl acids by H2CrO4 or KMnO4. A selective oxidation to aldehyde, instead, can be achieved using pyridinium chlorochromate (PCC) in stoichiometric amounts in aprotic solvents such as dichloromethane. Catalytic oxidation, instead, requires metal catalysts which are expensive and toxic (Hunsen 2005). A sustainable alternative is the use of Laccase/TEMPO-mediator systems, that has been used for the biocatalytic conversion of alcohols to aldehydes (Díaz-Rodríguez et al. 2014).
Laccases (EC 188.8.131.52, benzenediol:oxygen oxidoreductases) are a well explored group of enzymes belonging to the multi-copper oxidase (MCO) family. Members of the MCO family contain four copper ions which are organized in two sites. The type 1 copper obtains one electron from the substrate, transferring it via a His-Cys-His motive to the trinuclear center. The trinuclear center contains one type 2 and two type 3 copper ions and this is the site where the oxygen reduces to water. In this reaction, four electrons from substrate molecules are transferred to one molecule of O2, reducing it to two molecules of water (Mot and Silaghi-Dumitrescu 2012). Unlike other enzymes, laccases can oxidize various substrates, such as phenols (Majcherczyk 1999), aromatic and aliphatic amines and some inorganic ions, while reducing oxygen to water (Riva 2006). Plant laccases play an important role in the synthesis of lignin, while fungal laccases catalyze its degradation for wood-decay, pathogenesis, fungal morphology and detoxification (Zhao et al. 2013). These natural activities are exploited in several applications by different industrial processes. For example, in the food and textile industry, laccases are used to reduce the oxygen content, specifically in beer production to increase the product shelf-life and for their bleaching activities on denim fabrics, respectively (Mate and Alcalde 2017). Moreover, different studies showed the ability of laccases to produce polymeric structures (Braunschmid et al. 2021; Pollard and Bruns 2018).
Although laccases are widespread in nature, as they have been described from bacteria, fungi, higher plants and insects, fungal laccases represent the most significant group of the blue MCO family with regard to the number and extent of characterization. Typical fungal laccases are 60-70 kDa monomeric glycoproteins with a well characterized catalytic mechanism for the formation of radical species (Bassanini et al. 2020). Moreover, through the so-called “Laccase-mediator system” (LMS), radical species may play a role as mediators by oxidizing non-phenolic compounds. The catalytic cycle of laccases can only start if the substrate of interest has the proper redox potential. In fact, laccases typically possess a redox potential of about 0.5–0.8 mV vs. the normal hydrogen electrode (Witayakran and Ragauskas 2009). Due to steric hindrance and/or redox potential incompatibility, mediators can act as redox intermediates, following a nature mimicking-fashion strategy. The produced free radicals from the oxidation of these compounds can act on bulky or high redox potential substrates.
Since the discovery of 2,2'-azine-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) to be used as mediator, the number of compounds that has this function has dramatically increased (Morozova et al. 2007). In order to be considered a proper mediator, the redox compound must not inhibit the enzyme and its conversion must be cyclic. One of the most common mediators in LMSs is the compound 2,2,6,6-tetramethyl-1-piperidinyloxyl (TEMPO), which has sufficiently high redox potential and it is more efficient than other mediators. The TEMPO mediator is present in the solution in the form of a relatively stable N-oxyl radical which is also able to oxidize non-phenolic structures (Fabbrini et al. 2002). Shortly, laccases oxidize TEMPO to produce the oxo-ammonium ion, which reacts with the substrate. Proton removal yields the oxidized product and the reduced (N–OH) form of TEMPO. The reduced TEMPO is converted by laccases to the oxidized form and then to the oxo-ammonium ion (Morozova et al. 2007). The LMS offers the possibility to convert primary and secondary alcohols into oxidized compounds, such as aldehydes, acids and ketones.
The application of enzymes in synthetic chemistry requires the use of non-conventional reaction systems to dissolve hydrophobic substrates in the presence of water, including the use of organic solvents (Bassanini et al. 2020). Both water-miscible solvents (Wan et al. 2010) and biphasic water-immiscible solvents (Nicotra et al. 2004; Ponzoni et al. 2007) have been applied to improve substrate solubility.
Utilization of by-products and residues from food and agricultural industries as raw materials for laccase production permits a more sustainable process, both economically and environmentally.
In the present work, we highlight the comparative activity of crude and purified laccase-containing mixtures produced from four edible mushrooms which were grown on SSF using wheat straw as substrate, on the model reaction of the oxidation of benzyl alcohol to benzaldehyde through the laccase/TEMPO LMS with a biphasic system.