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.
Kotake
T
T
Department of Environmental Studies, Faculty of Integrated Arts & Sciences, Hiroshima University, Japan.
Nakagawa
N
N
Takeda
K
K
Sakurai
N
N
D000595:Amino Acid Sequence; D002473:Cell Wall / Q000201:enzymology; D002845:Chromatography; D043326:Glucan 1,3-beta-Glucosidase; D001467:Hordeum / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D001617:beta-Glucosidase; D043326:Glucan 1,3-beta-Glucosidase