Laccases can catalyze the remediation of hazardous synthetic dyes in an eco-friendly manner, and thermostable laccases are advantageous to treat high-temperature dyeing wastewater. A novel laccase from Geothermobacter hydrogeniphilus (Ghlac) was cloned and expressed in Escherichia coli. Ghlac containing 263 residues was characterized as a functional laccase of the DUF152 family. By structural and biochemical analyses, the conserved residues H78, C119, and H136 were identified to bind with one copper atom to fulfill the laccase activity. In order to make it more suitable for industrial use, Ghlac variant Mut2 with enhanced thermostability was designed. The half-lives of Mut2 at 50 °C and 60 °C were 80.6 h and 9.8 h, respectively. Mut2 was stable at pH values ranging from 4.0 to 8.0 and showed a high tolerance for organic solvents such as ethanol, acetone, and dimethyl sulfoxide. In addition, Mut2 decolorized approximately 100% of 100 mg/L of malachite green dye in 3 h at 70 °C. Furthermore, Mut2 eliminated the toxicity of malachite green to bacteria and Zea mays. In summary, the thermostable laccase Ghlac Mut2 could effectively decolorize and detoxify malachite green at high temperatures, showing great potential to remediate the dyeing wastewater.

Cellobiose phosphorylase (CBP) catalyzes the reversible phosphorolysis of cellobiose into α-glucose 1-phosphate and glucose. A CBP with a broadened substrate specificity would be more desirable when utilized to convert cellulose into amylose (PNAS, 110: 7182–7187, 2013) and to construct yeast that can phosphorolytically use cellodextrin to produce ethanol. Based on the structure differences in the catalytic loops of CBP and cellodextrin phosphorylase from Clostridium thermocellum (named CtCBP and CtCDP, respectively), CtCBP was mutated to change its substrate specificity. A single-site mutant S497G was identified to exhibit a 5.7-fold higher catalytic efficiency with cellotriose as a substrate in the phosphorolytic reaction compared to the wild type, without any loss of catalytic efficiency on its natural substrate, cellobiose. When the S497G variant was used in the transformation of mixed cellodextrin (cellobiose + cellotriose) to amylose, the amylose yield was significantly increased compared to that of wild-type CtCBP. A structure change in the substrate-binding pocket of the S497G variant accounted for its capacity to accept longer cellodextrins than cellobiose. Taken together, the modified CtCBP, S497G was confirmed to acquire a promising feature favorable to those application scenarios involving cellodextrin’s phosphorolysis.

The enzymatic hydrolysis of lignocellulose is often hindered by the glucose-mediated inhibition of β-glucosidases, a major bottleneck in industrial cellulose degradation. Identifying novel glucose-tolerant β-glucosidases is essential for enhancing saccharification efficiency. In this study, we cloned and heterologously expressed a novel β-glucosidase, RpBgl8, from the termite Reticulitermes perilucifugus in Escherichia coli. Sequence and structural analyses classified RpBgl8 as a glycoside hydrolase family 1 enzyme. The purified enzyme exhibited optimal activity at 45 °C and pH 7.0, with broad stability across pH 4.0–8.0. Notably, RpBgl8 demonstrated high tolerance to lignocellulose-derived inhibitors and organic solvents, maintaining 100% activity in 15% ethanol. Furthermore, RpBgl8 exhibited outstanding glucose tolerance, retaining 100% activity at 2.5 M glucose and 82% activity at 4.0 M glucose—outperforming most previously reported β-glucosidases. A structural analysis revealed a narrow, hydrophobic substrate pocket, with residue F124 at the glycone-binding site critical for minimizing glucose accumulation. The F124W mutation significantly reduced glucose tolerance, confirming that hydrophobic interactions at the active site mitigate inhibition. These findings establish RpBgl8 as a promising candidate for high-solid biomass processing and simultaneous saccharification and fermentation applications, highlighting termites as underexplored sources of biocatalysts with unique industrial potential.

Inhibitors from lignocellulosic biomass have become the bottleneck of biorefinery development. Gluconobacter oxydans DSM2003 showed a high performance of inhibitors degradation, which had a short lag time in non-detoxified corn stover hydrolysate and could convert 90% of aldehyde inhibitors to weaker toxic acids. In this study, an aldehyde dehydrogenase gene W826-RS0111485, which plays an important function in the conversion of aldehyde inhibitors in Gluconobacter oxydans DSM2003, was identified. W826-RS0111485 was found by protein profiling, then a series of enzymatic properties were determined and were heterologously expressed in E. coli. The results indicated that NADP is the most suitable cofactor of the enzyme when aldehyde inhibitor is the substrate, and it had the highest oxidation activity to furfural among several aldehyde inhibitors. Under the optimal reaction conditions (50 °C, pH 7.5), the Km and Vmax of the enzyme under furfural stress were 2.45 and 80.97, respectively, and the Kcat was 232.22 min⁻¹. The biodetoxification performance experiments showed that the recombinant E. coli containing the target gene completely converted 1 g/L furfural to furoic acid within 8 h, while the control E. coli only converted 18% furfural within 8 h. It was further demonstrated that W826-RS0111485 played an important role in the detoxification of furfural. The mining of this inhibitor degradation gene could provide a theoretical basis for rational modification of industrial strains to enhance its capacity of inhibitor degradation in the future.

The effective deconstruction of lignocellulosic biomass is essential for sustainable biorefineries. In this study, corn stover was pretreated by low-alkali (1–5 wt% NaOH) pre-impregnation assisted instant catapult steam explosion (ICSE) to investigate its influence on enzymatic hydrolysis efficiency and the mechanism of lignin-derived anti-enzymatic factors. The results showed that this pretreatment effectively enhanced glucose yield. Under 4–5% NaOH conditions, washed samples achieved glucose yields above 98%. At 4% NaOH, the glucose yields of washed and unwashed groups were 98.88% and 56.34%, respectively, indicating that washing removed soluble inhibitors. LC-MS analysis identified three major water-soluble inhibitory compounds-vanillin, syringaldehyde, and 2-carboxybenzaldehyde-confirming their negative effects on cellulase activity. The alkali-soluble lignin content of unwashed samples (43.28%) was 1.36 times higher than that of washed samples (31.93%), demonstrating its role as a water-insoluble inhibitory factor. Moreover, SEM, XRD, FTIR, and contact angle analyses revealed that 5% NaOH treatment enhanced lignin solubilization, induced structural rearrangement and interfacial hydrophilic reconstruction, and increased cellulose crystallinity and enzyme accessibility. These findings elucidate the mechanistic pathways of lignin transformation and inhibition mitigation, providing valuable insights for efficient and sustainable biomass conversion.

The effective deconstruction of lignocellulosic biomass is essential for sustainable biorefineries. In this study, corn stover was pretreated by low-alkali (1-5 wt% NaOH) pre-impregnation assisted instant catapult steam explosion (ICSE) to investigate its influence on enzymatic hydrolysis efficiency and the mechanism of lignin-derived anti-enzymatic factors. The results showed that this pretreatment effectively enhanced glucose yield. Under 4–5% NaOH conditions, washed samples achieved glucose yields above 98%. At 4% NaOH, the glucose yields of washed and unwashed groups were 98.88% and 56.34%, respectively, indicating that washing removed soluble inhibitors. LC-MS analysis identified three major water-soluble inhibitory compounds-vanillin, syringaldehyde, and 2-carboxybenzaldehyde-confirming their negative effects on cellulase activity. The alkali-soluble lignin content of unwashed samples (43.28%) was 1.36 times higher than that of washed samples (31.93%), demonstrating its role as a water-insoluble inhibitory factor. Moreover, SEM, XRD, FTIR, and contact angle analyses revealed that 5% NaOH treatment enhanced lignin solubilization, induced structural rearrangement and interfacial hydrophilic reconstruction, and increased cellulose crystallinity and enzyme accessibility. These findings elucidate the mechanistic pathways of lignin transformation and inhibition mitigation, providing valuable insights for efficient and sustainable biomass conversion.