Biochemical characterization and molecular dynamics study of a thermostable endo-1,5-α-arabinanase

Abstract

Arabinan, a crucial constituent of plant biomass, undergoes hydrolysis through the enzymes endo-1,5-α-L-arabinanases and α-L-arabinofuranosidases, resulting in the release of L-arabinose. This monosaccharide holds diverse applications in the biofuel, food, and pharmaceutical industries. In this study, we characterized GeoARA, a recombinant enzyme of endo-1,5-α-L-arabinanase from Geobacillus sp. JS12, previously undescribed in this organism. GeoARA, expressed in E. coli BL21 and purified via affinity chromatography, displayed optimal activity at pH 7.0 and temperature of 70 °C on the specific substrate debranched arabinan. In the presence of metallic ions and EDTA as additives, the enzyme demonstrated high stability. Notably, the enzyme maintained high thermal stability at 70°C for up to 48 h, with enhanced performance in the presence of Co²⁺. GeoARA demonstrated a specific activity on debranched arabinan of 226.7 U mg⁻¹, with K_m, V_max, k_cat, and k_cat/K_m values of 12.1 mg mL⁻¹, 1284.7 μmol min⁻¹ mg⁻¹, 642.3 s⁻¹, and 53 mL mg⁻¹ s⁻¹, respectively. Furthermore, the three-dimensional structure of GeoARA was determined using homology-based molecular modeling, and the predicted model quality was validated through molecular dynamics simulations. The integration of computational and biochemical analyses confirmed that the enhanced thermostability observed experimentally, particularly in the presence of the cobalt ion, is associated with reduced atomic fluctuations and increased structural rigidity near the catalytic site. Together, these biochemical features position the endo-1,5-α-L-arabinanase GeoARA as a promising candidate for efficiently extracting L-arabinose in industrial applications.