Purification and characterization of a novel extracellular o-1,3-glucanase complex (GluGgt) secreted by Gaeumannomyces graminis var. tritici

摘要

o-1,3-Glucanases produced by the take-all disease pathogen Gaeumannomyces graminis var. tritici (Ggt) have been suggested to be implicated in infection and colonization process of the pathogen in wheat. For further studying the role of these enzymes in pathogenesis by cytochemical technique, an extracellular o-1,3-glucanase complex (GluGgt), was purified from culture filtrate of Ggt by (NH)SO precipitation, hydrophobic-interaction, anion-exchange and size-exclusion chromatography. The complex consisted of two interconvertible isoforms (Ia and Ib), both active proteins Ia and Ib appeared to be glycosylated in native polyacrylamide gel electrophoresis (PAGE). The pI of the active proteins Ia and Ib determined by IEF-PAGE were 6.3 and 6.4, respectively. GluGgt yielded two subunits with molecular masses of 66.2 and 56.0 kDa, respectively, in 15% SDS-PAGE. The complex had a pH optimum of 5.0 and the optimal temperature was 50pC. The enzyme complex proved to be stable at a temperature up to 60pC and retained an optimal high activity in the pH range from 4.0 to 7.0. Enzyme activity of GluGgt was obviously stimulated by Mnpo ions and moderately inhibited in the presence of Hgpo and Copo ions and KMnO. The K m of GluGgt was estimated to be 1.08 mg mlp# for laminarin as substrate and the V max 0.20 omol minp#.