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

Abstract

β-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 β-1,3-glucanase complex (GluGgt), was purified from culture filtrate of Ggt by (NH4)2SO4 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 50°C. The enzyme complex proved to be stable at a temperature up to 60°C and retained an optimal high activity in the pH range from 4.0 to 7.0. Enzyme activity of GluGgt was obviously stimulated by Mn2+ ions and moderately inhibited in the presence of Hg2+ and Co2+ ions and KMnO4. The Km of GluGgt was estimated to be 1.08 mg ml−1 for laminarin as substrate and the Vmax 0.20 μmol min−1.