AN EXTRACELLULAR ALPHA-L-ARABINOFURANOSIDASE SECRETED FROM CELL-SUSPENSION CULTURES OF CARROT

Carrot (Daucus carota L. cv. Kintoki) cell cultures secrete an alpha-L -arabinofuranosidase (alpha-L-AFase, EC 3.2.1.55) into their culture m edium during growth. The extracellular alpha-L-AFase (alpha-L-AFase-II ) was purified to electrophoretic homogeneity from the concentrated me dium using ammonium sulfate precipitation, chromatography on DEAE-Seph arose CL-6B, CM-Sepharose CL-6B, Sephacryl S-200HR and Concanavalin A- Sepharose, and preparative PAGE. The molecular mass of the purified en zyme was estimated to be 84 kDa by Sephacryl S-200HR gel-permeation, a nd 80 kDa by SDS-PAGE under denaturing conditions. The enzyme containe d carbohydrate and protein in a ratio of 1:5 (w/w), and was analyzed f or 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 degrees C. The activity was inhibit ed by Zn2+, Ag2+, Cu2+, Hg2+ and p-chloromercuribenzoate. The K-m and V-max values for p-nitrophenyl-alpha-L-arabinofuranoside were 0.22 mM and 0.11 mmol (mg protein)(-1) h(-1), respectively. The enzyme acted o n beet arabinan in an exo-fashion, and was capable of hydrolysing arab inose-rich polymers purified from pectic polysaccharides of carrot cel l cultures. However, even after an exhaustive reaction, the enzyme had little or no effect on cell walls from carrot cell cultures.