Heterologous expression and characterization of a GH9 glucanase gene IDSGLUC9-25 from rumen microbiota in Hu sheep

Abstract:

【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 the IDSGLUC9-25 gene from the rumen fluid cDNA of Hu sheep and heterologously expressed it in Escherichia 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 °C and pH 6.0. The enzyme displayed robust catalytic activity within the temperature range of 30–50 °C. 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 from Treponema 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.