Non-redundant horse gut potential CAZYmes set.

Herbivores represent crucial subjects for mining highly efficient carbohydrate-active enzymes (CAZymes) from gut microbiomes. Here, we analyzed 12,763 metagenome-assembled genomes (MAGs) from the horse gut, revealing that its microbial community as a rich reservoir of CAZymes diversity, with 5,204,848 glycoside hydrolases (GHs) and 4,596,191 glycosyl transferases (GTs) identified. Our findings demonstrate that Bacteroidota (5,479,287 CAZymes) and Bacillota_A (2,987,684 CAZymes) serve as the primary functional phyla for plant polysaccharide degradation.A total of 17,250 polysaccharide utilization loci (PULs) discovered in Bacteroides species. Through comparative genomic screening, a total of 12,373 hypothetical genes were predicted in PULs. These genes represent a putative novel reservoir of CAZymes. We randomly selected and identified a putative novel CAZyme, which encodes 452 amino acids and is designated H113. Our research has confirmed that H113 is a multifunctional metal enzyme (Zn2+ significantly enhancing its catalytic efficiency) capable of degrading α-1,4 and β-1,4 glycosidic bonds, exhibiting optimal activity at pH 4.8 and 35howimaltotriose: 82.2 U/mg; cellobiose: 80.6 U/mg). Phylogenetic analysis revealed H113 belongs to a conserved enzyme family with 1,866 identified homologs. This study not only provides a reference for efficient discovery of novel CAZymes but also offers valuable resources for developing novel biocatalysts.