A novel bifunctional type I α-l-arabinofuranosidase of family 43 glycoside hydrolase (BoGH43_35) from Bacteroides ovatus with endo-β-1,4-xylanase activity

Abstract

The gut bacterium Bacteroides ovatus harbors a diverse arsenal of glycoside hydrolases (GHs), which play pivotal roles in degrading dietary polysaccharides. In this study, we characterized a novel glycoside hydrolase from family 43 and subfamily 35 (BoGH43_35), cloned from B. ovatus. The 1956 bp gene was expressed in Escherichia coli BL21 (DE3), yielding a homogeneous soluble recombinant enzyme (∼74 kDa) upon purification through immobilized metal-ion affinity chromatography (IMAC). BoGH43_35 exhibited a remarkable specificity for arabinoxylans, with maximum catalytic activity (4.9 U mg⁻¹) against wheat arabinoxylan (low-viscosity), followed by 3.0 U mg⁻¹ against rye arabinoxylan (high viscosity) and beechwood xylan (2.3 U mg⁻¹). Optimal enzymatic performance was achieved at 37 °C and pH 7.0 having kinetic parameters of V_max 5.7 U mg⁻¹ and K_M calculated to be 2.7 mg mL⁻¹ for wheat arabinoxylan. Notably, BoGH43_35 retained stability within an acidic pH range (4–5) and displayed a half-life of 89 min at 30 °C. Protein thermal stability assays revealed a melting temperature (T_m) of 41.0 °C. Thin-layer chromatography (TLC) and ¹H NMR analyses of hydrolysed products confirmed the enzyme's dual functionality: an initial α-l-arabinofuranosidase (EC 3.2.1.55) activity, followed by an endo-β-1,4-xylanase (EC 3.2.1.8) activity, as evidenced by the release of xylooligosaccharides, including xylobiose and xylotriose, from xylans. Further structural analysis demonstrated BoGH43_35's ability to hydrolyze monosubstituted arabinofuranosyl residues from α-1,2- or α-1,3-linked arabinoxylan, confirming its type I α-l-arabinofuranosidase activity. This multifunctional enzyme holds potential in the valorization of hemicellulosic biomass and the production of prebiotic oligosaccharides and other biotechnological applications.