Purification and properties of a novel beta-galactosidase or exo-(1-->4)-beta-D-galactanase from the cotyledons of germinated Lupinus angustifolius L. seeds.

The main polysaccharide component of the thickened cell walls in the storage parenchyma of Lupinus angustifolius L. cotyledons is a linear (1-->4)-beta-linked D-galactan, which is mobilised after germination (L. A. Crawshaw and J.S.G Reid, 1984, Planta 160, 449-454). The isolation from the germinated cotyledons of a beta-D-galactosidase or exo-(1-->4)-beta-D-galactanase with a high specificity for the lupin galactan is described. The enzyme, purified using diethylaminoethyl-cellulose, carboxymethyl-cellulose and affinity chromatography on lactose-agarose, gave two bands (major 60 kDa, minor 45 kDa) on sodium dodecyl sulphate-gel electrophoresis, and two similar bands on isoelectric focusing (major, pI 7.0, minor pI 6.7, both apparently possessing enzyme activity). The minor component cross-reacted with an antiserum raised against, and affinity-purified on, the major band. Both components had a common N-terminal sequence. The minor component was probably a degradation product of the major one. The enzyme had limited beta-galactosidase action, catalysing the hydrolysis of p-nitrophenyl-beta-D-galactopyranoside and (1-->4)- and (1-->6)-beta-linked galactobioses. Lactose [beta-D-galactopyranosyl-(1-->4)-D-glucose] was hydrolysed only very slowly and methyl-beta-D-galactopyranoside not at all. Lupin galactan was hydrolysed rapidly and extensively to galactose, whereas other cell-wall polysaccharides (xyloglucan and arabinogalactan) with terminal non-reducing beta-D-galactopyranosyl residues were not substrates. A linear (1-->4)-beta-linked galactopentaose was hydrolysed efficiently to the tetraose plus galactose, but further sequential removals of galactose to give the tetraose and lower homologues occurred more slowly. Galactose, gamma-galactonolactone and Cu+2 were inhibitory. No endo-beta-D-galactanase activity was detected in lupin cotyledonary extracts, whereas exo-galactanase activity varied pari passu with galactan mobilisation. Exo-galactanase protein was detected, by Western immunoblotting of cotyledon extracts, just before the activity could be assayed and then increased and decreased in step with the enzyme activity. The exo-galactanase is clearly a key enzyme in galactan mobilisation and may be the sole activity involved in depolymerising the dominant (1-->4)-beta-galactan component of the cell wall.