BsEndo9: A Thermostable GH9 Endoglucanase with Broad Substrate Specificity from Bacillus safensis

The exploitation of lignocellulosic biomass, driven by cellulolytic enzymes, offers a sustainable route for converting plant-based waste into biofuels, biochemicals, and other value-added products.

BsEndo9 is a novel glycoside hydrolase family 9 (GH9) endoglucanase identified in Bacillus safensis ATHUBA63, a soil-derived strain from the Attica region of Greece. The gene encoding BsEndo9 was amplified via PCR and heterologously expressed in E. coli BL21 (DE3) competent cells. The recombinant enzyme was purified using His-Tag-assisted chromatography and its molecular weight (69 kDa) was confirmed via SDS-PAGE. BsEndo9 exhibited optimal activity at 60 °C and pH 6.0, retaining over 85% of activity after 48 hours within the pH range 5.0–8.0. Thermal inactivation studies revealed a half-life of 74.53 min at 60 °C and an inactivation energy (E_(a)d) of 198.51 kJ/mol. Additional thermodynamic parameters (ΔH*, ΔS*, ΔG*) were also determined. Enzyme activity was enhanced by Mg²⁺ and stable in the presence of several metal ions (K⁺, Na⁺, Fe²⁺, Ca²⁺, Ba²⁺, Co²⁺, Mn²⁺, Ni²⁺, Zn²⁺), as well as EDTA and SDS, though inhibited by 5 mM Fe³⁺ and Cu²⁺. Substrate specificity was examined against amorphous cellulose substrates: carboxymethyl cellulose (CMC), β-glucan (barley) and phosphoric acid-swollen cellulose (PASC). Kinetic parameters (K_m and V_max) were determined for carboxymethyl cellulose (CMC). Structural prediction using AlphaFold3 (beta) indicated a modular structure with a GH9 catalytic domain and a CBM3 module, consistent with efficient cellulases.