Isolation of cellotriosyl blocks from barley β-glucan with endo-1,4-β-glucanase from Trichoderma reesei

Mixed-linkage β-glucan from barley, with a cellotriosyl to cellotetraosyl ratio of 2.9, was hydrolysed with two endo-1,4-β-glucanases (cellulases) and one non-cellulolytic β-glucanase isolated from Trichoderma reesei. The hydrolysates were precipitated in 90% ethanol and the fragments obtained were further treated with lichenase, followed by analysis of the oligosaccharides released. One of the endo-1,4-β glucanases, Cel 5A (EG II), selectively degraded cellotetraosyl units in the polymer and left the cellotriosyl units unhydrolysed. Blocks of cellotriosyl units were isolated on a larger scale using this enzyme. The isolated blocks were fractionated into four fractions using gel permeation chromatography on Biogel P-6 and the structures of the blocks were analysed by 1H NMR spectroscopy. The fractions essentially contained cellotriosyl units of different sizes with a 3-linked glucose residue at the non-reducing end and a reducing end linked to two 4-linked glucose residues. The results thus indicated that the enzyme could hydrolyse a 4-linked glucose residue next to the 3-linked residue at the non-reducing terminal but left two 4-linked glucose residues at the reducing end. The isolated blocks of cellotriosyl units had a molecular weight distribution that fits a theoretical model based on random blocks of triosyl units in the mixed-linkage β-glucan.