Molecular cloning and characterization of a GH10 thermophilic xylanase from hot spring and its potential application in promoting probiotic growth

Xylan is widely found in plant cell walls, and xylanase, an essential enzyme in xylan breakdown, has promising applications in energy, food, feed, and healthcare sectors. This study presents the discovery of a novel GH10 family xylanase gene, termed Lc-Xyn81, isolated from the hot spring of Eryuan, Dali, Yunnan Province, employing enrichment culture and metagenomic approaches. The amino acid sequence of Lc-Xyn81 shares 72.29% identity with that of Blastocatellia bacterium. The gene was amplified via specific PCR, cloned, and heterologously expressed in Escherichia coli. The recombinant Lc-Xyn81 was purified using Ni-affinity chromatography, followed by enzymatic characterization. Lc-Xyn81 demonstrated optimal activity at 75 °C and pH 6.6. It maintained over 80% relative activity between 65 and 75 °C, and its activity increased to over 120% after incubation at 70 °C for 40–100 min with a half-life of 180 min at 70 °C. Additionally, incubation at pH 5.0–7.0 for 12 h boosted its activity to over 140%. Lc-Xyn81 was activated by divalent metal ions such as Co²⁺ (128.55%), Mn²⁺ (119.84%), and Cu²⁺ (112.27%). The enzyme exhibited activity against beechwood xylan (213.68 U/mg), corncob xylan (143.40 U/mg), and sugarcane bagasse xylan (80.39 U/mg). The primary degradation products were xylobiose and xylotetraose, which significantly promoted the growth of L. lactis. Kinetic analysis indicated that the Km value of Lc-Xyn81 for beechwood xylan is 4.62 mg/mL, and its Vmax value is 312.5 µmol/min/mg. In summary, Lc-Xyn81, a thermophilic and thermostable xylanase, exhibits considerable potential for industrial applications in lignocellulose degradation and prebiotic production.