A Thermostable Multifunctional GH5 Endoglucanase From Bacillus sp. for Lignocellulosic Biomass Saccharification

Characterization of a Novel GH3 β-Glucosidase with Alkali-Tolerant and Halophilic Properties from Pontixanthobacter luteolus SW-109.

High-cell-density fermentation of Komagataella phaffii enables the overproduction of a novel, broad-substrate endoglucanase, WcEG394 from Fuling (Wolfiporia cocos), for lignocellulose biorefining.

Recombinant WcEG394 efficiently hydrolyzed carboxymethyl cellulose, microcrystalline cellulose, and filter paper, and demonstrated broad substrate specificity toward xylans, pectin, chitin, agarose and agricultural residues such as corn cob and sugarcane bagasse.

A new thermophilic Parageobacillus sp. DSM 35475 producing a thermostable hemicellulolytic secretome from spent mushroom substrate and digestate.

The development of robust enzyme systems and the use of low-cost feedstocks for lignocellulosic biomass deconstruction remain major challenges for biorefineries. Here, we report a strategy to discover and produce thermostable hemicellulolytic enzymes from industrial microbiomes enriched on spent mushroom substrate (SMS) at 70 °C. A thermophilic bacterium representing a putative novel species, Parageobacillus sp. DSM 35475, was isolated and cultivated on a medium composed exclusively of SMS and digestate, demonstrating the feasibility of producing extracellular enzymes from agro-industrial residues. The resulting cell-free secretome exhibited xylanase activity of 0.22 U/mL, with an optimum at 80 °C and pH 7, while retaining more than 30% of its maximal activity between 60 and 90 °C and more than 50% activity after prolonged incubation at 75-80 °C. More than 40% of the initial activity was preserved after 80 days of storage at 4 °C. HPAEC-PAD analysis of hydrolysis products, together with zymographic and genomic analyses, indicated the presence of a multifunctional hemicellulolytic system comprising endo-xylanase, β-xylosidase, α-L-arabinofuranosidase, and endo-mannanase activities. The secretome hydrolysed beechwood xylan, arabinoxylan and alkali-pretreated SMS, releasing mixtures of mono- and oligosaccharides under thermophilic conditions. Genome annotation predicted a diverse CAZyme repertoire and a dedicated xylan/arabinan utilisation locus consistent with the observed biochemical phenotype. Overall, this study demonstrates the feasibility of producing a thermostable hemicellulolytic secretome from low-cost agro-industrial residues and identifies Parageobacillus sp. DSM 35475 as a promising source of thermophilic enzymes for future lignocellulose bioconversion.

Enzymatic Characterization of a Novel GH3 β-Glucosidase from Lentilactobacillus buchneri: EDTA-Mediated Enhancement of Thermostability

Background/Objectives: β-Glucosidases from lactic acid bacteria are valuable biocatalysts for carbohydrate conversion, flavour enhancement, and bioactive glycoside biotransformation. Although GH3 family enzymes have been characterized from several Lactobacillus species, no systematic study exists for Lentilactobacillus buchneri—a GRAS strain with plant-polysaccharide-degrading potential. This work aimed to clone, heterologously express, and comprehensively characterize a novel GH3 β-glucosidase (LbBgl3) from L. buchneri, with a particular focus on its catalytic properties, substrate profile, stability, and the unexpected EDTA-mediated thermostabilization mechanism. Methods: A novel β-glucosidase gene (LbBgl3) from L. buchneri was successfully expressed in Escherichia coli. Results: Biochemical characterization revealed that LbBgl3 is a cold-adapted, moderately acidophilic enzyme, exhibiting optimal activity at 37 °C and pH 5.0, with a maximum specific activity of 799.67 U·mg−1. Under optimal conditions, the enzyme displayed kinetic parameters toward pNPG with a Km of 1.697 mM, Vmax of 442.5 μmol·mg−1·min−1, kcat of 634.32 s−1, and kcat/Km of 373.79 mM−1·s−1. LbBgl3 exhibited high salt tolerance, with activity peaking at ~140% at 0.5 M NaCl and retaining ~68% at 2.0 M NaCl, while showing sensitivity to glucose inhibition. Notably, EDTA significantly enhanced both the activity and stability of LbBgl3. At 50 mM, the relative activity increased to 161%, and stability at 25 °C was prolonged. Molecular docking simulations suggested that EDTA binds near the substrate-binding pocket, forming hydrogen bonds with Ala57, Gln766, Ser768, and Lys770, thereby stabilizing the local conformation and enhancing thermal resistance. Conclusions: This study presents the first characterization of a GH3 β-glucosidase from L. buchneri and reveals a non-classical stabilizing effect of EDTA, offering valuable insights for enzyme engineering and biocatalytic applications.

Efficient high-temperature simultaneous saccharification and fermentation of lignocellulosic substrates enabled by engineering thermotolerance in an enzyme-secreting industrial Saccharomyces cerevisiae strain.

The high cost of commercial enzyme cocktails remains a major barrier for lignocellulosic (second-generation) bioethanol production. Simultaneous saccharification and fermentation (SSF) at elevated temperatures using enzyme-secreting yeast can reduce enzyme demand, but is constrained by the limited thermotolerance of industrial strains. In this study, thermotolerant isolates of an inhibitor-tolerant, xylose-utilizing, enzyme-secreting industrial Saccharomyces cerevisiae strain were generated using whole-genome transformation (WGT). Screening in mixed-sugar fermentations at 41 °C identified several improved isolates, of which one isolate, designated Cellusec®4.0, achieved an ethanol titer of 5.45%(v/v), representing an 86% increase compared to the parental strain. This was driven by near-complete utilization of glucose, xylose, and cellobiose. In SSF at 40 °C with sorghum pulp, Cellusec®4.0 reached 5.83%(v/v) ethanol, 24% higher than the parental strain. Fed-batch SSF of pretreated softwood demonstrated the benefit of elevated temperature, with Cellusec®4.0 achieving 4.36%(v/v) ethanol at 40 °C, 29% higher than at 35 °C. In addition, fed-batch SSF of alkali-pretreated sugarcane bagasse at 39 °C using an in-house produced enzyme cocktail resulted in ethanol titers of up to 8.0% (v/v) within 48 h, corresponding to an 83% yield. These results demonstrate that WGT is an effective strategy to introduce thermotolerance into industrial yeast while maintaining key traits. The improved thermotolerance of Cellusec® 4.0 enabled high-temperature SSF, thereby increasing ethanol titers. Combined with retained inhibitor tolerance, enzyme secretion, and mixed-sugar utilization, this supported efficient ethanol production across multiple lignocellulosic substrates under industrially relevant conditions.

A highly efficient thermostable xylanase from Thermomyces dupontii J22: purification, properties and its application in xylan film modification