A new thermophilic Parageobacillus sp. DSM 35475 produces 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 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.