Purification and properties of wall-bound endo-1, 4-beta-glucanase from suspension-cultured poplar cells.

A wall-bound endo-1, 4-beta-glucanase (EC 3.2.1.4) was obtained from a preparation of the cell walls of suspension-cultured poplar cells and purified to electrophoretic homogeneity by cation-exchange, hydrophobic, and gel-filtration chromatography. The molecular mass was estimated to be 47 kDa by SDS-PAGE and 48 kDa by gel filtration on Superdex 200 pg. The isoelectric point (pI) was 5.6. The purified enzyme catalyzed the endo-hydrolysis of carboxymethylcellulose with an optimal pH of 6.5, a Km of 1.2 mg ml-1, and a Vmax of 280 units. The purified enzyme specifically hydrolyzed the 1, 4-beta-glucosyl linkages of carboxymethylcellulose, phospho-swollen cellulose, lichenan, xylan and xyloglucan. The activity of the enzyme was strongly stimulated by cysteine-HCl. The N-terminal sequence of the enzyme was similar to that of an extracellular endo-1, 4-beta-glucanase found in suspension cultures of poplar cells and some homology was recognized to avocado fruit-ripening and bean abscission endo-1, 4-beta-glucanases.