Purification and properties of a novel xyloglucan-specific endo-(1----4)-beta-D-glucanase from germinated nasturtium seeds (Tropaeolum majus L.).
Endo-(1----4)-beta-D-glucanase activity has previously been detected in the cotyledons of germinated nasturtium (Tropaeolum majus L.) seeds, and has been linked to the hydrolysis in vivo of storage xyloglucan (amyloid) (Edwards, M., Dea, I. C. M., Bulpin, P. V., and Reid, J. S. G. (1985) Planta (Berl.) 163, 133-140). Extracts from the cotyledons of 14-day seedlings are now shown to contain a single endo-glucanase activity, and it has been purified to apparent homogeneity by sequential anion-exchange chromatography, cation-exchange chromatography, and gel filtration. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis, dodecyl sulfate gel electrophoresis, and isoelectric focusing. The isoelectric point was 5.0, the pH optimum 4.5-5.0, and the temperature optimum 40 degrees C. Dodecyl sulfate electrophoresis gave a molecular weight of 29,000; permeation methods gave lower values. The enzyme contained about 5% carbohydrate. An endo mode of action on tamarind seed xyloglucan was deduced by monitoring solution viscosity and reducing power, and by analyzing the end products of the reaction. The specificity of the enzyme toward a wide range of substrates, including celluloses, carboxymethyl and hydroxyethyl celluloses, glucomannan, galactoglucomannans, mixed-linkage (1----3 and 1----4)-beta-D-glucan, laminarin, and xyloglucans from seeds and primary cell walls, was tested. Only the xyloglucans were hydrolyzed. It is concluded that the enzyme is a pure, endo-acting (1----4)-beta-D-glucanase which is novel in its apparently complete specificity toward xyloglucans. It is speculated that enzymes of this type may have escaped earlier detection because it is normal practice to screen for endo-(1----4)-beta-D-glucanase using artificial cellulose derivatives and that they may be widely involved in xyloglucan metabolism in plant cell walls.
D002480:Cellulase / Q000302:isolation & purification / Q000378:metabolism*; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D005936:Glucans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology*; D011134:Polysaccharides / Q000378:metabolism*; D012639:Seeds / Q000201:enzymology; D013379:Substrate Specificity; D013696:Temperature; D014990:Xylans
D005936:Glucans; D011134:Polysaccharides; D014990:Xylans; C029353:xyloglucan; D002480:Cellulase