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Family GH1 glycosyl hydrolases are ubiquitous in prokaryotes and eukaryotes and are utilized in numerous industrial applications, including bioconversion of lignocelluloses. In this study, hyperacidophilic archaeon Cuniculiplasma divulgatum (S5T=JCM 30642T) was explored as a source of novel carbohydrate-active enzymes. The genome of C. divulgatum encodes three GH1 enzyme candidates, from which CIB12 and CIB13 were heterologously expressed and characterized. Phylogenetic analysis of CIB12 and CIB13 clustered them with β-glucosidases from genuinely thermophilic archaea including Thermoplasma acidophilum, Picrophilus torridus, Sulfolobus solfataricus, Pyrococcus furiosus, and Thermococcus kodakarensis. Purified enzymes showed maximal activities at pH 4.5-6.0 (CIB12) and 4.5-5.5 (CIB13) with optimal temperatures at 50°C, suggesting a high-temperature origin of Cuniculiplasma spp. ancestors. Crystal structures of both enzymes revealed a classical (α/β)8 TIM-barrel fold with the active site located inside the barrel close to the C-termini of β-strands including the catalytic residues Glu204 and Glu388 (CIB12), and Glu204 and Glu385 (CIB13). Both enzymes preferred cellobiose over lactose as substrates and were classified as cellobiohydrolases. Cellobiose addition increased the biomass yield of Cuniculiplasma cultures growing on peptides by 50%, suggesting that the cellobiohydrolases expand the carbon substrate range and hence environmental fitness of Cuniculiplasma., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

A mesophilic, hyperacidophilic archaeon, strain M1 T , was isolated from a rock sample from Vulcano Island, Italy. Cells of this organism were cocci with an average diameter of 1 µm. Some cells possessed filaments. The strain grew in the range of temperatures between 15 and 52 °C and pH 0.5-4.0 with growth optima at 40 °C and pH 1.0. Strain M1 T was aerobic and chemoorganotrophic, growing on complex substrates, such as casamino acids, trypticase, tryptone, yeast and beef extracts. No growth at expenses of oxidation of elemental sulphur or reduced sulphur compounds, pyrite, or ferrous sulphate was observed. The core lipids were glycerol dibiphytanyl glycerol tetraether lipids (membrane spanning) with 0 to 4 cyclopentane moieties and archaeol, with trace amounts of hydroxy archaeol. The dominant quinone was MK-7 : 7. The genome size of M1 T was 1.67 Mbp with a G+C content of 39.76 mol%, and both characteristics were well within the common range for Thermoplasmatales . The phylogenetic analysis based on 16S rRNA gene sequence placed the strain M1 T within the order Thermoplasmatales with sequence identities of 90.9, 90.3 and 90.5% to the closest SSU rRNA gene sequences from organisms with validly published names, Thermoplasma acidophilum , Thermoplasma volcanium and Thermogymnomonas acidicola , respectively. Based on the results of our genomic, phylogenetic, physiological and chemotaxonomic studies, we propose that strain M1 T (=DSM 116605 T =JCM 36570 T ) represents a new genus and species, Oxyplasma meridianum gen. nov., sp. nov., within the order Thermoplasmatales .