Heterologous Expression, and Characterization of a Thermophilic and Acid-Tolerant GH10 Xylanase from Thermomonospora amylolytica

Xylanases, a subclass of glycoside hydrolases, catalyze the breakdown of xylan into xylan disaccharides, oligosaccharides, and xylose, playing a critical role in the degradation and utilization of hemicelluloses. However, extreme conditions hinder their broader industrial applications. In this study, a GH10 xylanase gene (referred to as taxyn10a) was sourced from Thermomonsospora amylolytica, cloned, and heterologously expressed in Escherichia coli BL21(DE3). The recombinant TaXyn10A was purified using Ni2+-affinity chromatography. Its optimal temperature and pH were determined to be 65°C and 5.6, respectively. The enzyme maintained over 80% of its activity between 55-70°C, with about 60% residual activity after 120 minutes at 60°C. Additionally, it preserved more than 80% of its activity in a pH range of 3.6-6.6. TaXyn10A exhibited over 60% activity in the presence of 1 or 10 mM ions (K+, Fe3+, Mg2+, Ca2+, Zn2+, Mn2+, Ni2+, Co2+, Pb2+, Cu2+), as well as 1% EDTA, PMSF, and DTT. Kinetic analysis with beechwood xylan yielded apparent Vmax, Km, and kcat values of 69.44 μmol min⁻¹ mg⁻¹, 24.53 mg mL⁻¹, and 53.35 s⁻¹, respectively. These findings demonstrate that TaXyn10A was active over moderately acidic pH and elevated-temperature ranges and tolerates various metal ions and chemical additives, highlighting its potential for feed processing and lignocellulosic biomass conversion.