TL;DR
Local enrichment of acidic residues on the PlGH3 surface could generate a negative electrostatic potential, which enables adaptation to high-salt and alkaline environments, thereby sustaining the enzyme's catalytic activity under such extreme conditions.
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.
β-glucosidases belong to the class of glycoside hydrolases (GHs) and can mediate the cleavage of β-1-4 glycosidic bonds in alkyl or aryl-β-D-glucosides, amino, cyanogenic glucosides, and oligo- or disaccharides. The gene encoding β-glucosidase of Anoxybacillus flavithermus DSM2641T was subcloned and expressed in E. coli BL21 Star(DE3) cells with pET100 Directional TOPO Expression Kit. The recombinant AFBGL was purified by a Ni-NTA affinity chromatography. The enzyme was 53 kDa and had 5.13 × 105 µmol/min/mg protein of Vmax and 422.3 µM of Km towards p-nitrophenyl β-D-glucoside as a main substrate. The optimum temperature and pH of the purified enzyme were 65°C and 6.8, respectively. We also determined kinetic parameters of AFBGL against the other artificial substrats, pNPC, pNPX, and oNPG. The enzyme had the Vmax and Km for 5.13 × 105 µmol/min/mg protein and 0.42 mM for pNPG, 0.007 µmol/min/mg protein and 0.19 mM for pNPC, 0.01 µmol/min/mg protein and 11.97 mM for pNPX, 0.06 µmol/min/mg protein and 0.134 mM for oNPG, respectively. AFBGL's substrate affinity was found to be more directed towards oNPG. Although 0.5 mM Mg2+ increases the enzyme's activity 1.6-fold, higher concentrations of Mg2+ and the presence of K+, Fe2+, Mn2+, Zn2+, Cu2+, and Al3+ decreases its activity. AFBGL retained 50% of the initial activity towards pNPG for 25 min and the activity was not detected after 225 min. According to amino acid motifs, AFBGL belongs to family 1 of glycosylhydrolase and 4/7 super family. So, AFBGL is a member of GH1, as the amino acid sequence contained a GH1 specific domain.
BSEG2 is highly thermostable, tolerant of high salt and ionic liquids, and notably resistant to cellobiose inhibition up to 200 mM, suggesting potential to reduce freshwater demand in biomass processing and a strong candidate for simplified, cost-effective enzyme formulations for glucose production from lignocellulosic feedstocks.
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.
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.