Characterization of beta-Glucosidase Isoenzymes Possibly Involved in Lignification from Chick Pea (Cicer arietinum L.) Cell Suspension Cultures

Abstract

Crude cell wall preparations from Cicer arietinum L. cell suspension cultures show high activity for the hydrolysis of coniferyl alcohol β-d-glucoside (coniferin). Various β-glucosidase activities could be solubilized from these preparations by 0.5 M NaCl treatment and one of these could be shown to possess a high activity for the hydrolysis of coniferin. The enzyme activities were purified to near homogeneity by Sephadex G-200 and CM-Sephadex chromatography. Isoelectric focussing indicated the occurrence of β-glucosidase isoenzymes with identical catalytic activity (pI 8.5–10). Molecular weights were determined as 110 000, with two subunits of 63000 and 43000. Maximum hydrolytic activity is at pH 5. The β-glucosidase isoenzymes catalyze the hydrolysis of various β-glucosides with aromatic aglycone structure and different sugar moieties. However, coniferin has been found to be one of the best substrates (k_m= 0.8 mM; V= 6 μmol · min⁻¹· mg protein⁻¹) for these β-glucosidase isoenzymes. The data suggest that a β-glucosidase-catalyzed reaction might be involved in lignification of these plant cell cultures.