Lignocellulosic biomass has been generally recognized to be an abundant and renewable feedstock for sustainable energy production, yet its efficient bioconversion remains technically a big challenge. Among it, enzymatic saccharification is the central step that links feedstock pretreatment to the downstream fermentation [1], [2], which relies on the concerted action of three classes of cellulases, involving endoglucanases (EC 3.2.1.4), exoglucanases (EC 3.2.1.91), and β-glucosidases (EC 3.2.1.21) [3]. Notably, the endoglucanases play a pivotal role by randomly cleaving internal β-1,4-glycosidic bonds in cellulose chains, thereby generating new chain ends for subsequent exoglucanase action [4]. Thus, catalytic performance of these endoglucanases directly influences the efficiency of cellulose hydrolysis.

Commercial cellulase preparations consisting of endoglucanases are predominantly derived from mesophilic fungi such as Trichoderma spp. [5] and Penicillium spp. [6]. While these enzymes are widely used, they are prone to a rapid inactivation at elevated temperatures, resulting in poor catalytic efficiency and reduced economic feasibility under industrial conditions. In contrast, thermophilic fungi and bacteria produce endoglucanases with a higher thermal stability, enabling the hydrolysis at elevated temperatures where the reaction rates are fast, the risk of microbial contamination is reduced, and substrate accessibility is improved [7]. In particular, thermophilic bacteria, i.e., Bacillus, Clostridium, and Thermotoga, have emerged as a potent candidate for thermostable cellulases that can be readily expressed in prokaryotic systems like Escherichia coli [8], [9]. For instance, an endoglucanase (TnCelB) from Thermotoga neapolitana is optimally active at 90 °C [10]. Recently, a GH5 endoglucanase CelA from Dictyoglomus turgidum (DtCelA) has gained attention, owing to its broad substrate spectrum and high reaction temperature. Despite these advantages, DtCelA showed a limited thermostability above 75 °C, retaining approximately 30 % of optimal activity at 90 °C, and displayed relatively low specific activity (63 U/mg) toward CMC substrate [11]. These shortcomings have impeded the applicability in ligonocellulosic biorefinery development, which underscores the need for further improvement of both catalytic activity and thermal stability.

Currently, the improvement in enzyme activity and stability is primarily achieved through enzyme engineering including rational design and carbohydrate binding module (CBM) fusion [12]. In recent years, structure-based site-directed mutagenesis has become an effective tool in rational enzyme engineering, as it targets the enzyme’s active center or key structural sites by replacing specific amino acids to enhance enzymatic properties [13]. Zuo et al. obtained two dominant mutants by site-directed mutation of key amino acid residues in the catalytic pocket of Oenococcus oeni β-glucosidase, the activities of which were increased by 2.81 and 3.18 times respectively compared to the wild type [14]. Disulfide bond engineering can also be achieved through site-directed mutagenesis, which enhances the protein thermostability by introducing intramolecular disulfide bonds [15]. Ding et al. introduced a disulfide bond into glucose 1-dehydrogenase (LsGDH) derived from Lysinibacillus sphaericus G10, generating mutant DS255 with a half-life of 9900 min at 50 °C, which is 1868 times longer than that of the wild type [16]. On the other hand, the performance of enzyme proteins has been found to improve by fusion with a class of non-catalytic proteins, CBMs [17], [18]. CBMs can target catalytic domains to the substrate surface and increase enzyme concentration there, thereby enhancing substrate specificity [19] [20]. Sajjad et al. fused the structural domain of CBM3a with the endoglucanase CelA derived from the thermophilic bacterium Clostridium thermocellum, thereby generating a series of fusion enzymes and increasing the activities of CelA-BC and CelA-CBC on pretreated sugarcane bagasse by nearly 3-fold and 5-fold, respectively, compared to the original enzyme CelA-CD [21]. However, it is often difficult for a single strategy to simultaneously enhance enzyme activity and stability, whereas the synergistic effect of multiple strategies may be implementable in the overall enzymatic performance.

In this study, we optimized the enzymatic properties of DtCelA, a GH5 family endoglucanase derived from the thermophilic bacterium Dictyoglomus turgidum. Unlike conventional single-strategy approaches, this study firstly applied a cascade of three strategies for DtCelA modification to achieve synergistic optimization of enzymatic properties such as activity, stability, and substrate binding ability. Firstly, a targeted mutation engineering approach based on homology modeling and molecular docking was employed to enhance its enzymatic activity. To further improve thermal stability, disulfide bonds were introduced in conjunction with the aforementioned strategy to reinforce protein rigidity. Finally, the mutant was fused with a carbohydrate-binding module (CBM) to boost catalytic efficiency and balance the relationship between enzyme activity and thermal stability. By integrating these three optimization strategies, this study provided a theoretical foundation for the development of industrial cellulase preparation.