Cellulosomal AcXyn30B_12 from Acetivibrio clariflavus acts synergistically with AcGH30A upon the hydrolysis of complex carbohydrates.

AcGH30A and AcXyn30B_12 are two of the most abundant enzymes in the cellulosome of the thermophilic anaerobe Acetivibrio clariflavus. Their surprising abundance within the glycolytic repertoire of this highly efficient microorganism, active in sewage sludge ecosystems, suggests a cooperative role in the hydrolysis of complex carbohydrates. Here, we cloned, expressed and characterized the endo/exo-β-1,4-xylanase AcXyn30B_12, which has a molecular weight of ~74 kDa and displays optimal activity at pH 5.5 and 70 °C. AcXyn30B_12 exhibited broad substrate specificity, with the highest catalytic efficiency against partially acetylated birchwood xylan (PABX), yielding a V of 133.3 U/mg and a K of 0.9 mg/mL. AcXyn30B_12 activity was enhanced by Ca (10 %) and Mg (7.3 %) ions. The enzyme also showed notable thermostability and pH tolerance, maintaining activity up to 60 °C and within a pH range of 4.5-8.0. Time-course hydrolysis experiments revealed the ability of AcXyn30B_12 to release a variety of xylo-oligosaccharides (from xylopentaose to xylobiose) and xylose from PABX, confirming its endo-acting mechanism. Additionally, AcXyn30B_12 effectively degraded lignocellulosic biomass, releasing significant amounts of xylo-oligosaccharides and xylose from complex substrates. In contrast, AcGH30A, previously characterized as an exo-xylobiohydrolase, removes xylobiose from non-reducing ends of xylan and xylo-oligosaccharides. Our experiments demonstrate the synergistic action of AcGH30A and AcXyn30B_12 in complex carbohydrate hydrolysis, with AcXyn30B_12 generating the non-reducing ends that serve as substrates for AcGH30A. This enzyme synergy underscores the potential industrial applications of these enzymes in high-temperature processes, including prebiotic production, bioethanol generation and the paper and pulp industries.