Expression and characterization of a novel microbial GH9 glucanase, IDSGLUC9-4, isolated from sheep rumen

• Published in: Animal bioscience 2024, 37(9), , pp.1581-1594
• Main Authors: Zhu, Yongzhen, Bai, Shuning, Li, Nuo, Wang, Jun-Hong, Wang, Jia-Kun, Wang, Qian, Wang, Kaiying, Zhang, Tietao
• Format: Article
• Language: English
• Subjects: expression; glycoside hydrolase; hydrolysis; rumen microbiome; β-glucan; 축산학
• ISSN: 2765-0189; 2765-0235
• DOI: 10.5713/ab.24.0138

Objective: This study aimed to identify and characterize a novel endo-β-glucanase, IDSGLUC9-4, from the rumen metatranscriptome of Hu sheep.Methods: A novel endo-β-glucanase, IDSGLUC9-4, was heterologously expressed in Escherichia coli and biochemically characterized. The optimal temperature and pH of recombinant IDSGLUC9-4 were determined. Subsequently, substrate specificity of the enzyme was assessed using mixed-linked glucans including barley β-glucan and Icelandic moss lichenan. Thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), matrix assisted laser desorption ionization time of flight mass spectrometry analyses were conducted to determine the products released from polysaccharides and cello-oligosaccharides substrates.Results: The recombinant IDSGLUC9-4 exhibited temperature and pH optima of 40°C and pH 6.0, respectively. It exclusively hydrolyzed mixed-linked glucans, with significant activity observed for barley β-glucan (109.59±3.61 μmol/mg min) and Icelandic moss lichenan (35.35±1.55 μmol/mg min). TLC and HPLC analyses revealed that IDSGLUC9-4 primarily released cellobiose, cellotriose, and cellotetraose from polysaccharide substrates. Furthermore, after 48 h of reaction, IDSGLUC9-4 removed most of the glucose, indicating transglycosylation activity alongside its endo-glucanase activity.Conclusion: The recombinant IDSGLUC9-4 was a relatively acid-resistant, mesophilic endo-glucanase (EC 3.2.1.4) that hydrolyzed glucan-like substrates, generating predominantly G3 and G4 oligosaccharides, and which appeared to have glycosylation activity. These findings provided insights into the substrate specificity and product profiles of rumen-derived GH9 glucanases and contributed to the expanding knowledge of cellulolytic enzymes and novel herbivore rumen enzymes in general.