Characteristics of β‐galactosidase purified from cell suspension cultures of carrot

Abstract

Five glycosidase activities from cell homogenate of carrot ( Daucus carota L. cv. Kintoki) cell cultures were assayed after extraction successively by phosphate buffer (pH 7.0) and the buffer plus 2 M NaCl. A β‐galactosidase (EC 3.2.1.23) was isolated in a highly purified state from the buffer‐soluble protein fraction by ammonium sulfate fractionation and chromatography on CM‐Sephadex C‐50, DEAE‐Sephadex A‐50 and Sephadex G‐200. The molecular weight of this enzyme was ca 104 000 and the isoelectric point was pH 7.8. The optimal activity occurred at pH 4.4 with McIlvaine buffer. The K m and V max values were 1.67 m M and 201 units (mg protein) −1 , respectively, for p ‐nitrophenyl β‐ d ‐galactopyranoside. The enzyme activity was strongly inhibited by Zn 2+ , Cu 2+ , Hg 2+ and d ‐galactono‐1,4‐lactone. The enzyme acted on the β‐1,4‐linked galactan prepared from citrus pectin in an exo‐fashion. Furthermore, the enzyme was slightly involved in the hydrolysis of the pectic polymer and cell walls purified from carrot cell cultures.