Characteristics, hydrolysis of cell wall polymers, and response to calcium deficiency of a cell wall-associated β-galactosidase from carrot cells

The activity of β-galactosidase (EC 3.2.1.23) in the protein fraction solubilized with 3 mol/L LiCl from cell walls of suspension-cultured carrot (Daucus carota) cells has been found to be higher than those of the other glycosyl-hydrolytic enzymes. The cell wall-associated β-galactosidase/exo-galactanase was purified to electrophoretic homogeneity. Analysis by denaturing PAGE revealed a single polypeptide chain with a molecular mass of 50 kDa, similar to 52 kDa estimated for the native protein by size-exclusion chromatography. The enzyme contained carbohydrate and protein in a ratio of 1 : 10 (w/w), and was rich in Gly and Phe, followed by Ser, Ala and Thr. The isoelectric point was pH 6.5, and pH and temperature optima 4.4 and 45-50 °C, respectively. The β-galactosidase activity was inhibited by Mg2+, Ag+, Hg2+, p-chloromercuribenzoate and D-galactono-1,4-lactone. The Km and V max values for p-nitrophenyl-β-D-galactopyranoside were 0.77 mmol/L and 0.32 mmol (mg protein)−1 h−1, respectively. The enzyme hydrolyzed citrus galactan in an exo-fashion and was capable of hydrolysing an acidic pectic polymer containing galactosyl and arabinosyl residues from carrot cell walls and, therefore, is an exo-galactanase. However, even after an exhaustive reaction, the enzyme had no effect on a galactose-rich hemicellulosic polymer from carrot cell walls. Under calcium deficiency the activities of a cell wall-associated α-galactosidase (EC 3.2.1.22) and β-galactosidase increased until 15 days of culture and then decreased gradually, whereas β-glucosidase (EC 3.2.1.21) activity increased markedly with time despite poor cell growth.