Plant and Cell Physiology, 1997, Vol. 38, No. 2 194-200
© 1997
Research Paper |
Purification and Characterization of Wall-bound Exo-l,3-ß-D-Glucanase from Barley (Hordeum vulgare L.) Seedlings
1 Department of Environmental Studies. Faculty of Integrated Arts & Sciences, Hiroshima University Higashi-Hiroshima, 739 Japan
2 Research Institute for Bioresources, Okayama University Kurashiki, Okayama, 710 Japan
A ß-D-glucanase activity hydrolyzing 1,3:1,4-ß-D-glucan was released from the cell walls of barley by 3M 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 l,4-ß-D-glucosidase (56.5%). The purified ß-D-glucanase has a pH optimum at 5.0, and hydrolyzes oligosaccharides containing ß-D-1,3 or ß-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, l,3:l,4-ß-D-glucan (from barley), laminarin and pustulan are also hydrolyzed, but not carboxylmethyl-curdlan, carboxymethyl-cellulose, xyloglucan and maltose. The purified ß-D-glucanase yielded monomeric glucose from laminarihexaose, and exhibited characteristics of an exo-l,3-ß-D-glucanase (EC 3.2.1.58 [EC] ). The activity and biochemical characteristics of this enzyme suggest that it is an exo-l,3-ß-D-glucanase involved in the rapid turnover of l,3:l,4-ß-D-glucan in barley cell walls during seedling growth.
(Received September 24, 1996; Accepted December 9, 1996)
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