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

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: In summary, Lc-Xyn81, a thermophilic and thermostable xylanase, exhibits considerable potential for industrial applications in lignocellulose degradation and prebiotic production.