Yu_2025_Bioresour.Technol_443_133801

Xylan represents a promising resource for generating high-value bioproducts in lignocellulosic biorefineries. Nevertheless, the intricate structure of xylan poses challenges for its degradation, requiring the synergistic action of specialized side-chain cleaving enzymes to enhance the efficiency of endo-xylanases. In this study, we identified and characterized a novel acetylxylan esterase (BtAcXE) from Bacteroides thetaiotaomicron VPI-5482, featuring a unique dual-catalytic domain architecture comprising an N-terminal CE1 domain (NTD) and a C-terminal CE3 domain (CTD). BtAcXE exhibited remarkable activity of 6864.1 +/- 745.6 U/mg against p-nitrophenylacetate, exceeding that of the individual NTD and CTD enzymes, as well as an equimolar mixture of both domains. Furthermore, supplementing xylanase with BtAcXE enhanced the hydrolysis of birchwood xylan by 1.9-fold, yielding up to 0.182 +/- 0.003 g/g acetic acid and 0.304 +/- 0.029 g/g reducing sugars, respectively. Acetic acid yields of 0.019-0.058 g/g were further achieved from more complex pretreated lignocellulosic feedstocks. Notably, the native dual-domain structure of BtAcXE conferred inter-domain synergy, resulting in an overall increase in the release of both acetic acid and reducing sugars. This study establishes BtAcXE as a highly efficient Axe with substantial potential for enhancing the high-value utilization of lignocellulosic biomass and provides valuable insights for designing and applying multicatalytic-domain xylanolytic enzymes.