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

Abstract

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.

Keywords: Expression; Glycoside Hydrolase; Hydrolysis; Rumen Microbiome; β-Glucan.

Figure 1

Phylogenetic analysis of IDSGLUC9-4 (GenBank accession no. WUV41754.1). Multiple sequence alignment was performed and used to generate a phylogenetic tree using MEGA11.0 and the maximum likelihood (ML) statistical method based on the WAG correction model, with a bootstrap of 500. The tree was drawn to scale (length = 0.1), and branch length was measured according to the number of substitutions per site.

Figure 2

Multiple sequence alignment of IDSGLUC9-4 and six functionally-characterized GH 9 endoglucanases. The three catalytic residues and the conserved aromatic residues in the active site of GH9 glucanases are indicated by red and green asterisks, respectively. Cel9Q, from Clostridium thermocellum (PDB: 5GXX); Cel9A, from Alicyclobacillus acidocaldarius (PDB: 3EZ8); CelT, from Clostridium thermocellum (PDB: 2YIK); CelR, from Acetovibrio thermocellus (PDB: 7UNP); Cel9G, from Clostridium thermocellum (PDB: 1G87); Cel9I, from Clostridium thermocellum (PDB: 2XFG).

Figure 3

Purification and substrate selectivity determination of recombinant IDSGLUC9-4. (A) SDS-PAGE (M is standard protein marker, 1 is purified IDSGLUC9-4). Agar plates treated with BSA (1) and IDSGLUC9-4 (2), containing 0.2% polysaccharide substrate: barley β-glucan (B), Icelandic moss lichenan (C), xyloglucan (D), konjac gum (E). SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Figure 4

Enzymic properties of IDSGLUC9-4. (A) pH profile of enzyme activity. (B) pH stability. (C) Temperature profile of enzyme activity. (D) Thermostability. Data expressed as the mean±standard deviation (n = 3).

Figure 5

Hydrolytic products generated by IDSGLUC9-4 from glucan-like polysaccharides and cello-oligosaccharides analyzed by TLC. (A) barley β-glucan. (B) Icelandic moss lichenan. (C) Xyloglucan. (D) Cello-oligosaccharides. G1, glucose; G2, cellobiose; G3, cellotriose; G4, cellotetraose; G5, cellopentaose. TLC, thin-layer chromatography.

Figure 6

HPLC and MALDI-TOF analyses of the hydrolytic products of IDSGLUC9-4. (A) HPLC profiles (B); reducing sugar profile; (C) MALDI-TOF analysis at 3 h; (D) MALDI-TOF analysis at 48 h of hydrolyzed barley β-glucan. (E) HPLC profiles; (F) reducing sugar profile; (G) MALDI-TOF analysis at 1h; (H) MALDI-TOF analysis at 48 h of hydrolyzed Icelandic moss lichenan. Data expressed as mean±standard deviation (n = 3). G1, glucose; G2, cellobiose; G3, cellotriose; G4, cellotetraose; G5, cellopentaose. HPLC, high-performance liquid chromatography; MALDI-TOF, matrix assisted laser desorption ionization time of flight mass spectrometry.

Figure 7

HPLC analyses of the hydrolytic product time-courses of IDSGLUC9-4. (A–E) HPLC profiles of G5 (A), G4 (B), G3 (C), G2 (D), and G1 (E). (A’–E’) Hydrolytic product profiles of G5 (A’), G4 (B’), G3 (C’), G2 (D’), and G1 (E’). Data are expressed as mean±standard deviation (n = 3). G1, glucose; G2, cellobiose; G3, cellotriose; G4, cellotetraose; G5, cellopentaose. HPLC, high-performance liquid chromatography.