applied sciences Review Mushroom Ligninolytic Enzymes—Features and Application of Potential Enzymes for Conversion of Lignin into Bio-Based Chemicals and Materials Seonghun Kim 1,2 1 Jeonbuk Branch Institute, Korea Research Institute of Bioscience and Biotechnology, 181 Ipsin-gil, Jeongeup 56212, Korea; [email protected]; Tel.: +82-63-570-5113 2 Department of Biosystems and Bioengineering, KRIBB School of Biotechnology, University of Science and Technology (UST), 217 Gajeong-ro, Daejeon 34113, Korea Abstract: Mushroom ligninolytic enzymes are attractive biocatalysts that can degrade lignin through oxido-reduction. Laccase, lignin peroxidase, manganese peroxidase, and versatile peroxidase are the main enzymes that depolymerize highly complex lignin structures containing aromatic or aliphatic moieties and oxidize the subunits of monolignol associated with oxidizing agents. Among these enzymes, mushroom laccases are secreted glycoproteins, belonging to a polyphenol oxidase family, which have a powerful oxidizing capability that catalyzes the modification of lignin using synthetic or natural mediators by radical mechanisms via lignin bond cleavage. The high redox potential laccase within mediators can catalyze the oxidation of a wide range of substrates and the polymeriza- tion of lignin derivatives for value-added chemicals and materials. The chemoenzymatic process using mushroom laccases has been applied effectively for lignin utilization and the degradation of recalcitrant chemicals as an eco-friendly technology. Laccase-mediated grafting has also been Citation: Kim, S. Mushroom employed to modify lignin and other polymers to obtain novel functional groups able to conjugate Ligninolytic Enzymes—Features and small and macro-biomolecules. In this review, the biochemical features of mushroom ligninolytic Application of Potential Enzymes for enzymes and their potential applications in catalytic reactions involving lignin and its derivatives to Conversion of Lignin into Bio-Based obtain value-added chemicals and novel materials in lignin valorization are discussed. Chemicals and Materials. Appl. Sci. 2021, 11, 6161. https://doi.org/ 10.3390/app11136161 Keywords: lignin; ligninolytic enzyme; mushroom; laccase; lignin peroxidase; manganese peroxidase; versatile peroxidase; lignin valorization; lignin bioconversion; laccase-mediated grafting Academic Editor: Alejandro Rodriguez Pascual Received: 14 June 2021 1. Introduction Accepted: 30 June 2021 Lignocellulose is a renewable bioresource used to produce bio-based fuel, chemicals, Published: 2 July 2021 and materials. Lignocellulosic biomass is an abundant and renewable resource from plants such as soft grasses, hard woods, agricultural crops, and their waste byproducts, mainly Publisher’s Note: MDPI stays neutral composed of polysaccharides. These plant cell walls consist of a complex polyphenolic with regard to jurisdictional claims in lignin combined with rigid cellulose amorphous hemicellulose structures [1,2]. Since plant published maps and institutional affil- cell walls are arranged with multi-layers consisting of lignin, cellulose, and hemicellulose, iations. which have strong interactions with each other, the disturbance and breakdown of poly- meric structures are the initial processes required for the utilization of subunit compounds such as sugar monomers, oligomers, and other aromatic derivatives [1,2]. Lignin binds to cellulose fibers to harden and strengthen plant cell walls. Cellulose is the main structural Copyright: © 2021 by the author. polysaccharide of the primary plant cell wall, and it accounts for 30–50% of the dry weight Licensee MDPI, Basel, Switzerland. of lignocellulose. The second polysaccharide component of lignocellulose is hemicellulose, This article is an open access article which accounts for 15–34% of the plant cell wall. The third main component is lignin. Con- distributed under the terms and ceptually, the microbial bioconversion of renewable chemicals from the biomass involves conditions of the Creative Commons four main steps: physical and chemical pretreatment of biomass, depolymerization of the Attribution (CC BY) license (https:// pretreated biomass, fermentation of the resulting sugars, and purification or distillation of creativecommons.org/licenses/by/ the products [3,4]. Other processes directly depolymerize biomass under harsh physical 4.0/). Appl. Sci. 2021, 11, 6161. https://doi.org/10.3390/app11136161 https://www.mdpi.com/journal/applsci Appl. Sci. 2021, 11, 6161 2 of 34 and chemical reactions, and the lysed monomers or oligomers can then be converted to renewable products through chemical or biological catalysts [1–4]. Theoretically, the main three fractions (% dry wt), cellulose (30–50%), hemicellulose (15–34%), and lignin (9–32%), from renewable sources could be converted completely to fuels and chemicals within the concept of net-zero emissions technology [5]. Nevertheless, different physical or chemical pretreatment procedures are employed for various types of lignocellulosic biomass, which may influence the downstream process for the conversion efficiency and productivity of final products [5,6]. Over recent decades, the process of the extraction of cellulose and hemicellulose linked to lignin from different types of biomass has been well-established to obtain fer- mentable sugars for the production of cellulosic biofuels or bio-building-block substances for chemical polymers [1,2,4]. Since cellulose and hemicellulose consist of simple or uni- form monosaccharide subunits, these fractions can be extracted easily as monomeric or oligomeric sugars through pretreatment processes rather than lignin-containing complex heteropolymers within aromatic structures [6–8]. Most of the conventional pretreatments comprised chemical methods combined with physical processes to enhance the cellulose content per gram of biomass or to extract hemicellulose fractions [6–11]. However, lignin fractions are released as byproducts of these pretreatment processes [7,10,12]. The high content lignin substances are then found in the wastewater generated by the pretreat- ment processes. Treatment of this wastewater accounted for approximately 20% of overall production costs [13]. The extracted lignin could be potentially utilizable for renewable chemicals to reduce non-utilized materials from biomass and overall production costs down for a sustainable green technology [14]. Lignin consists of aromatic heteropolymers with complex chemical linkages. The heteropolymeric structures contain hydroxyl, methoxyl, carbonyl, and carboxyl groups linked to aromatic or aliphatic moieties, with different molecular weights [15]. Lignin valorization is challenging due to the complexity of its structures compared with other cellulose and hemicellulose polymers [15,16]. The chemical bonds from the heterogeneous structures in lignin should be cleaved off by sophisticated catalysts with chemo-, regio-, and stereo-selectivity. Therefore, ligninolytic enzymes could be potential biocatalysts to convert lignin into valuable aromatic compounds [17–19]. Although plant lignin is resistant to biodegradation, many microorganisms, including fungi, secrete lignocellulotic enzymes to decay biomass for mineralization of the recalcitrant compounds [19]. These microor- ganisms, along with other degraders in nature, play a carbon-recycling role in ecosystems. These bacterial degradation ecosystems could potentially mimic lignin conversion into high-value aromatic compounds [16,17,19]. Ligninolytic enzymes that could be used for lignin valorization (i.e., that can selec- tively carry out regio- and stereo-specific bond formation under mild reaction conditions) have been highlighted as a promising method to overcome the limitations associated with classical chemical catalysts [17,18]. In this review, I describe the biochemical proper- ties of ligninolytic enzymes reported from mushroom species and their applications for chemoenzymatic approaches to produce renewable aromatic compound derivatives. 2. Structure of Lignin for Valuable Aromatic Compounds Lignin is one of the main components consisting of a plant biomass. Lignin has a unique structure of closely associated cellulose and hemicellulose, which resists the bio-degradation of plant cell walls to protect plants against environmental stress [19–22]. Lignin is a heterogeneous polymer containing various aromatic structures consisting of phenylpropane subunits, linked with carbon–carbon (C–C) or ether (C–O–C) into a complex three-dimensional structure [20–22]. The complex structure of lignin is initially biosynthe- sized from an aromatic amino acid phenylalanine or tyrosine. Then, the aromatic amino acid is converted into p-coumaryl alcohol via the phenylpropanoid pathway following deamination, ring hydroxylation, methylation, and carboxyl reduction. Following that, p-coumaryl alcohol (H-unit) is elaborated to coniferyl and sinapyl alcohols (G- and S-units). Appl. Sci. 2021, 11, x FOR PEER REVIEW 3 of 31 Appl. Sci. 2021, 11, 6161 3 of 34 coumaryl alcohol (H-unit) is elaborated to coniferyl and sinapyl alcohols (G- and S-units). TheseThese phytochemical phytochemical substances ofof thethe three three alcohol alcohol types types are are called called monolignols monolignols or lignols,or lig- nols,which which are the are main the main building building blocks blocks of lignin of lignin structures structures [23–25 [23] (Figure–25] (Figure1). 1). FigureFigure 1. 1. SchematicSchematic representation representation of of biosynthesis biosynthesis