Histidine ammonia-lyase (HAL) plays a pivotal role in the non-oxidative deamination of L-histidine to produce trans-urocanic, a crucial process in amino acid metabolism. This study examines the cloning, purification, and biochemical characterization of a novel HAL from Geobacillus kaustophilus (GkHAL) and eight active site mutants to assess their effects on substrate binding, catalysis, thermostability, and secondary structure. The GkHAL enzyme was successfully overexpressed and purified to homogeneity. Its primary sequence displayed 40.7% to 43.7% similarity with other known HALs and shared the same oligomeric structure in solution. Kinetic assays showed that GkHAL has optimal activity at 85 °C and pH 8.5, with high thermal stability even after preincubation at high temperatures. Mutations at Y52, H82, N194, and E411 resulted in a complete loss of catalytic activity, underscoring their essential role in enzyme function, while mutations at residues Q274, R280, and F325 did not abolish activity but did reduce catalytic efficiency. Notably, mutants R280K and F325Y displayed novel activity with L-histidinamide, expanding the substrate specificity of HAL enzymes. Circular dichroism (CD) analysis showed minor secondary structure changes in the mutants but no significant effect on global GkHAL folding. These findings suggest that GkHAL could be a promising candidate for potential biotechnological applications.

Xylan, the principal component of hemicellulose, requires the concerted action of endo- and exo-acting xylanolytic enzymes for its complete depolymerization into fermentable sugars. This study reports the biochemical and thermodynamic characterization of a glycoside hydrolase family 10 endo-β-1,4-xylanase from the thermophilic bacterium Geobacillus stearothermophilus CECT43 (GsXynA), cloned and heterologously expressed in Escherichia coli BL21(DE3) and purified to homogeneity by immobilized metal-ion affinity chromatography. The 338-residue enzyme behaved as a monomer and showed maximal activity on xylan substrates at pH 6.5 and 75 °C, retaining over 90% residual activity between pH 5 and 9 and remaining stable up to 70 °C, after 60 min of incubation. Kinetic and thermodynamic analyses of thermal inactivation monitored by enzymatic activity measurements revealed an enthalpy-driven denaturation process, while circular dichroism indicated that this irreversible unfolding is kinetically, not thermodynamically, controlled. GsXynA hydrolyzed beechwood xylan mainly to xylobiose and xylotetraose, consistent with mixed endo and exo activity, and was uncompetitively inhibited by 4-hydroxybenzoic acid. Combined action with the β-xylosidase GsXynB2 markedly increased D-xylose release, reaching a 53.23% hydrolysis yield and a degree of synergy of 3.79 after 24 h. These results demonstrate that GsXynA is a thermostable, versatile xylanase with promising potential for lignocellulosic biomass bioconversion.