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 bacte-rium. The gene was amplified via specific PCR, cloned, and heterologously expressed in Esche-richia 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 65–75°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, incu-bation at pH 5.0–7.0 for 12 h boosted its activity to over 140%. Lc-Xyn81 was activated by di-valent metal ions such as Co²⁺(128.55%), Mn²⁺ (119.84%), and Cu²⁺(112.27%). The enzyme ex-hibited 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 and Vmax values for Lc-Xyn81 were 4.62 mg/ml and 312.5 µmol/min/mg, respectively. Conclusions In summary, Lc-Xyn81, a thermophilic and thermostable xylanase, exhibits considerable poten-tial for industrial applications in lignocellulose degradation and prebiotic production.