An extracellular α‐L‐arabinofuranosidase secreted from cell suspension cultures of carrot

Abstract

Carrot (Daucus carota L. cv. Kintoki) cell cultures secrete an α‐L‐arabinofuranosidase (α‐L‐AFase, EC 3.2.1.55) into their culture medium during growth. The extracellular α‐L‐AFase (α‐L‐AFase‐II) was purified to electrophoretic homogeneity from the concentrated medium using ammonium sulfate precipitation, chromatography on DEAE‐Sepharose CL‐6B, CM‐Sepharose CL‐6B, Sephacryl S‐200HR and Concanavalin A‐Sepharose, and preparative PAGE. The molecular mass of the purified enzyme was estimated to be 84 kDa by Sephacryl S‐200HR gel‐permeation, and 80 kDa by SDS‐PAGE under denaturing conditions. The enzyme contained carbohydrate and protein in a ratio of 1:5 (w/w), and was analyzed for amino acid composition and the sequence of the first 21 amino acids of the N‐terminus. The isoelectric point was pH 5.6, the pH optimum 3.8, and the temperature optimum 55°C. The activity was inhibited by Zn2+, Ag2+, Cu2+, Hg2+ and p‐chloromercuribenzoate. The Km and Vmax values for p‐nitrophenyl‐α‐L‐arabinofuranoside were 0.22 mM and 0.11 mmol (mg protein)−1 h−1, respectively. The enzyme acted on beet arabinan in an exo‐fashion, and was capable of hydrolysing arabinose‐rich polymers purified from pectic polysaccha‐rides of carrot cell cultures. However, even after an exhaustive reaction, the enzyme had little or no effect on cell walls from carrot cell cultures.