Premium
An extracellular β‐galactosidase secreted from cell suspension cultures of carrot. Its purification and involvement in cell wall‐polysaccharide hydrolysis
Author(s) -
Konno Haruyoshi,
Katoh Kenji
Publication year - 1992
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1992.tb05819.x
Subject(s) - chromatography , extracellular , sephadex , daucus carota , galactan , chemistry , cell wall , molecular mass , biochemistry , hydrolysis , ammonium sulfate precipitation , enzyme , polysaccharide , size exclusion chromatography , biology , botany
β‐Galactosidase (β‐Galase, EC 3.2.1.23) activity has been detected in a culture medium of cell suspension cultures of carrot ( Daucus carota L. cv. Kintoki). The extracellular β‐Galase (β‐Galase‐II) was purified to electrophoretic homogeneity from the concentrated medium using ammonium sulfate precipitation, chromatography on CM‐Sephadex C‐50. DEAE‐Sepharose CL‐6B and Sephacryl S‐200HR, and preparative PAGE. The molecular mass of the purified enzyme was estimated to be 65 kDa by Sephacryl S‐200HR gel‐permeation, and 60 kDa by SDS‐PAGE after treatment with SDS and 2‐mercaptoethanol. The pI was 6.5. The K m and V max values for p ‐nitrophenyl (PNP)‐β‐D‐galactopyranoside were 0.17 m M and 31.9 μmol (mg protein) ‐1 , h ‐1 , respectively. The optimal activity in McIlvaine's buffer occurred at pH 4.0–4.4. The enzyme activity was inhibited by Co 24 , Cu 2+ , Hg 2‐ , p ‐chloromercuribenzoate (PCMB) and D‐galactono‐1,4‐lactone. The enzyme acted on citrus galactan and larchwood arabinogalactan in an exo‐fashion, and was slightly involved in the hydrolysis of an acidic pectic polymer containing arabinosyl and galactosyl residues and in the breakdown of cell walls isolated from carrot cell cultures.