ヤギ反芻動物細菌メタゲノムからの相同性およびモチーフベースのアプローチを用いたエンドグルカナーゼ遺伝子の同定

The goat rumen harbors a dense and diverse microbial community that produces enzymes for degrading plant cell wall polysaccharides, representing a potential source of valuable enzymes for applications. In this study, we explored the diversity of endoglucanases from goat rumen bacterial metagenomes using homology- and motif-based approaches. HMM profiling identified 5,182 endoglucanase-encoding ORFs, whereas KEGG annotation (EC 3.2.1.4) detected 7,368 ORFs, with 2,902 shared between the two methods. Taxonomic assignment revealed Firmicutes and Bacteroidetes as the dominant phyla, followed by Fibrobacteres, Spirochaetes, and Lentisphaerae. At the genus level, Ruminococcus (Firmicutes) and Prevotella (Bacteroidetes) were dominant, followed by Butyrivibrio, Fibrobacter, and Treponema. Pfam domain analysis further revealed that the common endoglucanase domains, GH5 and GH9, exhibited a high degree of overlap (> 80%) between the two approaches. Notably, GH26 and GH8 were more frequently identified by HMM profiling, whereas KEGG-based mining revealed greater domain diversity. A candidate endoglucanase-encoding ORF was subsequently expressed in E. coli, and the recombinant protein was characterized. The enzyme displayed bifunctional activity on both cellulose (CMC, filter paper) and hemicellulose (xylan), with the highest activity toward CMC, and showed optimal activity at 50 °C and pH 4.0. Its activity was differentially affected by metal ions and chemical agents, and kinetic analysis revealed strong substrate affinity and high catalytic efficiency (Km = 0.8 mg/mL, Vmax = 186.12 U/mg). Overall, this study demonstrates complementary strategies for mining functional proteins from metagenomes and highlights the potential of goat rumen-derived endoglucanase for lignocellulose degradation in sustainable industrial applications.