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[Objective] Glucanases serve as one of the main components in feed additives. This study identified and characterized a novel GH9 glucanase gene derived from rumen microbiota in herbivores, aiming to provide a reference for the research and development of feed enzymes. [Methods] We obtained theIDSGLUC9-25 gene from the rumen fluid cDNA of Hu sheep and heterologously expressed it inEscherichia coli. The recombinant protein was induced for expression by isopropyl β-d-thiogalactopyranoside, purified, and then subjected to functional characterization. [Results] IDSGLUC9-25 encoded a protein consisting of 527 amino acid residues, which included a CelD_N domain and a GH9 family catalytic domain. The recombinant rIDSGLUC9-25 protein exhibited a molecular weight of approximately 62.7 kDa and the highest enzymatic activity at 40 ℃ and pH 6.0. The enzyme displayed robust catalytic activity within the temperature range of 30–50 ℃. After preincubation at pH 4.0–8.0 for 1 h, rIDSGLUC9-25 retained the relative activity over 90%. The substrate spectrum analysis revealed that rIDSGLUC9-25 exhibited specific activities against barley β-glucan, moss lichenan, konjac gum, and xyloglucan, with the activities of (443.55±24.48), (65.56±5.98), (122.37±2.85), and (159.16±7.73) U/mg, respectively. The hydrolysis assay showed that rIDSGLUC9-25 primarily catalyzed the hydrolysis of β-glucan into cellotriose (representing 64.19%±1.19% of total reducing sugars) and cellotetraose (representing 26.24%±0.12% of total reducing sugars). Additionally, the enzyme predominantly generated cellotriose from the hydrolysis of lichenan (representing 78.46%±0.89% of total reducing sugars). [Conclusion] This study characterizes IDSGLUC9-25, an endo-β-1,4-glucanase (EC 3.2.1.4) derived fromTreponema sp. The enzyme exhibited robust activity in the conversion of polysaccharides into cellotriose and cellotetraose, establishing a foundation for the development of feed enzymes and functional oligosaccharides preparation.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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