Extracellular exo‐polygalacturonase secreted from carrot cell cultures. Its purification and involvement in pectic polymer degradation

Abstract

Exo‐polygalacturonase (exo‐PGase, EC 3.2.1.67) activity has been detected in a culture filtrate of cell suspension cultures of carrot (Daucus carota L. cv. Kintoki). The extracellular exo‐PGase was purified to electrophoretic homogeneity using DEAE‐Sephadex A‐50 ion‐exchange chromatography, Sephadex G‐150 gel filtration, and preparative polyacrylamide gel electrophoresis (PAGE). The molecular mass of the purified enzyme was calculated to be 48 kDa from Sephadex G‐200 gel filtration, and 50 kDa from sodium dodecyl sulfate (SDS)‐PAGE after treatment with SDS and 2‐mercaptoethanol. The isoelectric point was at pH 6.2. The Km and Vmax values for polygalacturonate (degree of polymerization: 52) were 14.4 μM and 25.6 μmol (mg protein)−1 h−1, respectively. The optimal activity in McIlvaine's buffer occurred at pH 4.6. The enzyme activity was inhibited by Ba2+, Cu2+, Mn2+ and Hg2+. The enzyme was involved in ca 15% hydrolysis of the acidic polymer purified from carrot pectic polysaccharides, and connected with the release of galacturonic acid. Even after an exhaustive reaction the enzyme had, however, little or no effect on cell walls from carrot cell cultures.