Purification and characterization of wall-bound exo-1,3-beta-D-glucanase from barley (Hordeum vulgare L.) seedlings.

A beta-D-glucanase activity hydrolyzing 1,3:1,4-beta-D-glucan was released from the cell walls of barley by 3 M LiCl treatment. It was purified by sequential cation-exchange, gel-filtration and hydrophobic chromatography. The molecular mass of the glucanase was 66 kDa as determined by SDS-polyacrylamide gel electrophoresis. Sequence determination of the first thirty amino acids of the N-terminus revealed a high homology of this enzyme to the Pseudomonas 1,4-beta-D-glucosidase (56.5%). The purified beta-D-glucanase has a pH optimum at 5.0, and hydrolyzes oligosaccharides containing beta-D-1,3 or beta-D-1,4 linkage. The glucanase showed maximum hydrolytic activity toward laminaritetraose, the rate being about two times that of cellotetraose and about four times that of gentiobiose. Polysaccharides such as lichenan, 1,3:1,4-beta-D-glucan (from barley), laminarin and pustulan are also hydrolyzed, but not carboxylmethyl-curdlan, carboxymethyl-cellulose, xyloglucan and maltose. The purified beta-D-glucanase yielded monomeric glucose from laminarihexaose, and exhibited characteristics of an exo-1,3-beta-D-glucanase (EC 3.2.1.58). The activity and biochemical characteristics of this enzyme suggest that it is an exo-1,3-beta-D-glucanase involved in the rapid turnover of 1,3:1,4-beta-D-glucan in barley cell walls during seedling growth.