As one of the three major components (i.e. cellulose, hemicellulose, and lignin) in lignocellulosic biomass, lignin plays a central role in structurally supporting the rigidity of plant cell walls while providing protections against environmental stress [1], [2]. Although extensive studies have been conducted to investigate the utilization of lignin to generate value-added fuels, chemicals, and materials, lignin valorization remains challenging due to the natural recalcitrance and heterogeneity of this aromatic polymer [3], [4]. Therefore, it is remarkably important to develop cost-effective and sustainable technologies that can unlock lignin as a bioresource to reduce waste, generate valuable products, and support the transition to a circular economy.

Lignin depolymerization is critical in the process of lignin valorization, as this step is treated as an efficient approach to reduce lignin heterogeneity by lowering the molecular weight of lignin and cleaving the inter-unit linkages, resulting in relatively uniform lignin products for upgrading [5], [6]. Prior to lignin depolymerization, lignin fractionation from biomass is generally carried out to extract lignin from plant cell walls or fractionate lignin into different streams by disrupting the crosslinks between lignin and cellulose/hemicellulose [7]. Extensive biomass fractionation methods, including physical pretreatment, chemical pretreatment, physico-chemical pretreatment, thermal pretreatment, biological pretreatment, and non-conventional pretreatment (such as plasma, ultrasound, and high-pressure homogenization) have been investigated to effectively extract lignin from biomass [8]. Among different lignin fractionation methods, utilization of ionic liquids (ILs) and deep eutectic solvents (DESs) has emerged as a promising technique to selectively fractionate/solubilize lignin due to their unique properties such as biocompatibility, high chemical and thermal stability, and low volatility [9], [10], [11].

In the current biorefining industry, thermochemical conversion (such as pyrolysis, catalytic oxidation, and gasification) and biological conversion (including microorganisms and enzymes for degradation) are two major approaches to valorize lignin [12], [13], [14]. Unlike thermochemical conversion, which is typically an energy-intensive process, biological conversion generally happens in a milder and more selective way [13], [14]. Lignin-degrading enzymes, such as lignin peroxidase, manganese peroxidase, and laccase, have attracted increased consideration due to their rapid reaction rate and specificity compared to lignin-degrading microorganisms [15], [16].

Despite the great promise that lignin-degrading enzymes hold for lignin valorization, there are limitations associated with the applications of these enzymes, such as their complex structure, low-yield production, and inefficient purification [17]. Thus, it is critical to develop technologies to produce enzymes in a cost-effective and productive way, for instance, heterologous protein expression in model microorganisms [17], [18]. Moreover, the development of biocompatible ILs and DESs has created promising approaches to consolidate lignin fractionation with enzymatic lignin depolymerization [19], [20]. Consequently, it is crucial to understand the interactions between lignin-degrading enzymes and ILs/DESs and to enhance the efficiency, selectivity, and specificity of lignin-degrading enzymes.

Protein engineering emerges as a pivotal approach to enhance enzyme performance, with a particular emphasis in thermal and kinetic stability [21]. The stability of proteins is subjected to multifaceted factors, wherein interactions like hydrogen bonds and surface charges of the proteins play critical roles, especially in scenarios involving the use of ILs and DESs [22]. Various strategies encompassing rational design, directed evolution, and de novo synthesis are employed to strategically modify enzyme composition, thereby effectively mitigating unfavorable protein interactions [23], [24], [25]. This orchestrated effort culminates in elevated enzyme performance. Advanced technologies give rise to a convergence of computational and molecular methodologies, which are promising tools in protein engineering [26]. The advent of innovative computational tools coupled with state-of-the-art molecular techniques expeditiously propels the generation of engineered proteins, consequently conferring acceleration and cost-efficiency to the process.

This review elucidates recent research progress on enzymatic lignin depolymerization, specifically in a consolidated process involving ILs/DESs. Moreover, the interactions between lignin-degrading enzymes and ILs/DESs and potential approaches to improve the performance of lignin-degrading enzymes via protein engineering are discussed.