Purification of an α‐L‐arabinofuranosidase from carrot cell cultures and its involvement in arabinose‐rich polymer degradation

Abstract

Konno, H., Yamasaki, Y. and Katoh, K. 1987. Purification of an α‐L‐arabinofurano‐sidase from carrot cell cultures and its involvement in arabinose‐rich polymer degradation.An α‐L‐arabinofuranosidase (α‐L‐arabinofuranoside arabinofuranohydrolase, EC 3.2.1.55) was isolated from a homogenate of cell suspension cultures of carrot (Daucus carota L. cv. Kintoki). The buffer‐soluble enzyme was purified to homogeneity by a procedure involving ammonium sulfate fractionation, chromatography on DEAE‐Sephadex A‐50, Sephadex G‐150, Con A‐Sepharose 4B and CM‐Sephadex C‐50, and preparative polyacrylamide gel electrophoresis. The size of this enzyme as determined by polyacrylamide gel electrophoresis in the presence of sodium laurylsulfate and by Sephadex G‐200 gel filtration was 94 and 110 kDa, respectively. The isoelectric point was at pH 4.7. The Km and Vmax values for p‐nitrophenyl α‐L‐arabinofuranoside were 1.33 mM and 20.2 μimol (mg protein)‐1 h‐1, respectively. The optimal activity occurred at pH 4.2 with Mcllvaine buffer. The enzyme was stimulated by Ca2+ and Zn2+, whereas it was strongly inhibited by Cu2+, Ag2+, Hg2+, p‐chloromercuri‐benzoate and L‐arabono‐l,4‐lactone. The enzyme acted on beet arabinan in an exo‐fashion. Furthermore, the enzyme was partially involved in the hydrolysis of the ara‐binogalactan and pectic polymer purified from carrot cell walls.