Purification and Characterization of Wall-bound Exo-1,3-β-D-Glucanase from Barley (Hordeum vulgare L.) Seedlings

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A β-_D-glucanase activity hydrolyzing 1,3:1,4-β-_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-β-_D-glucosidase (56.5%). The purified β-_D-glucanase has a pH optimum at 5.0, and hydrolyzes oligo-saccharides containing β-_D-1,3 or β-_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-β-_D-glucan (from barley), laminarin and pustulan are also hydrolyzed, but not carboxylmethyl-curdlan, carboxymethyl-cellulose, xylo-glucan and maltose. The purified β-_D-glucanase yielded monomeric glucose from laminarihexaose, and exhibited characteristics of an exo-1,3-β-_D-glucanase (EC 3.2.1.58). The activity and biochemical characteristics of this enzyme suggest that it is an exo-1,3-β-_D-glucanase involved in the rapid turnover of 1,3: 1,4-β-_D-glucan in barley cell walls during seedling growth.
日本植物生理学会の論文