Metagenomic mining unveils a novel GH130 enzyme with exclusive xylanase activity over a wide temperature and pH ranges.

The equine gut harbors a diverse microbial community and represents a rich source of carbohydrate-active enzymes (CAZymes). To identify and characterize potentially novel CAZymes from a horse's hindgut metagenome, shotgun metagenomic sequencing was performed on DNA extracted from a stool sample of a male horse, followed by CAZyme annotation. Here, we report on the characterization of a novel enzyme (AH2) that was identified, synthesized, cloned, and characterized from the obtained CAZyme dataset. AH2 was identified as a GH130 family member and displayed exclusive xylanase activity, a trait hitherto unreported in prior characterization of GH130 CAZymes. AH2 displayed an optimal activity at a pH of 5.6 and a temperature of 50°C. AH2 maintained significant activity across a pH range of 4-10 (62-72%) and temperatures of 30-70°C (77-86%). The enzyme had remarkable stability, with minimal reductions in activity across a temperature range of 4-70°C and pH levels of 3, 7, and 9. Docking studies identified AH2's amino acids (Glu90 and Glu149) to be involved in substrate binding. Molecular dynamics simulation confirmed the structural stability of AH2 at pH 5.6 and 50°C, further supporting its resilience under these conditions. Our results expand on the known activities associated with the GH130 CAZyme family and demonstrate that the horse gut metagenome represents an unexplored source of novel CAZymes. ONE-SENTENCE SUMMARY: A novel activity for members of the CAZyme family GH130.