Comparison of (1 → 3)(1 → 4)-β- d-glucan-4-glucanohydrolases (E.C. 3.2.1.73) from Fibrobacter succinogenes and from Bacillus subtilis and Use of High-performance Anion Exchange Chromatography in Product Characterization

A Fibrobacter succinogenes β-glucanase cloned in Escherichia coli, as isolated from the fermentation medium and without further purification, hydrolyzed (1 → 3)(1 → 4)-β- d-glucan but was without activity against a wide variety of other polysaccharides. The major products of hydrolysis of oat β-glucan were 3-O-β-cellobiosyl- d-glucose and 3-O-β-cellotriosyl- d-glucose identified by methylation analysis. A novel method, using high-performance anion exchange chromatography with pulsed amperometric detection, is described for analysis of the oligosaccharide reaction products and for comparison of different enzyme actions. Oligosaccharides of degree of polymerization (DP) from 3-9, obtained after exhaustive hydrolysis by the F. succinogenes enzyme, were quantitatively identical to those produced by the known (1 → 3)(1 → 4)-β- d-glucan-4-glucanohydrolase, E.C. 3.2.1.73, of Bacillus subtilis. Partial hydrolysis products of considerably higher DP were also analyzed and were qualitatively the same with each enzyme. Despite the apparent similarity of mode of action, the F. succinogenes enzyme appeared to have greater difficulty than the B. subtilis enzyme in digesting the intact cell-wall substrate, particularly the aleurone cell wall. As a consequence, slightly lower values for β-glucan were found when the F. succinogenes enzyme was substituted for the B. subtilis enzyme in an assay for (1 → 3)(1 → 4)-β- d-glucan in flours from oats and barley.