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

## TL;DR

Scientists discovered and tested a new enzyme from sheep rumen that breaks down plant sugars into specific smaller units, which could be useful for biofuel or industrial applications.

## Contribution

The study reports the isolation, expression, and biochemical characterization of a novel GH9 glucanase from sheep rumen metatranscriptome.

## Key findings

- IDSGLUC9-4 has optimal activity at 40°C and pH 6.0, showing strong activity on barley β-glucan and lichenan.
- The enzyme primarily produces cellobiose, cellotriose, and cellotetraose from glucan substrates.
- IDSGLUC9-4 exhibits transglycosylation activity, removing glucose after prolonged reaction.

## Abstract

This study aimed to identify and characterize a novel endo-β-glucanase, IDSGLUC9-4, from the rumen metatranscriptome of Hu sheep.

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.

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.

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.