Purification and characterization of an endo-polygalacturonase (PG1) from a Zimbabwean species ofArmillaria

Abstract A major endo-polygalacturonase (PG1), produced both in vivo in inoculated potato tubers and in vitro on crude cell walls of Corylus avellana by an isolate of a Zimbabwean species of Armillaria (group III), was purified 18.8-fold, to apparent homogeneity on SDS-PAGE, by gel filtration followed by cation-exchange chromatography. PG1 was highly stable throughout the purification process, with no detectable loss of activity. A single band with a molecular weight of 40 kDa representing PG1 was obtained on silver-stained SDS-PAGE gels. No glycosyl groups could be detected by concanavalin A on blots after SDS-PAGE indicating the enzyme was probably not glycosylated. PG1 has an isoelectric point of 7.3 as indicated by activity staining and Coomassie blue staining of gels after isoelectric focusing. The enzyme showed activity over a broad pH range, with an optimum of pH 5.5 and a preference for polygalacturonic acid to pectin, with a polygalacturonic acid to pectin ratio of 1.4. Analysis of reaction products, obtained by incubating polygalacturonic acid with PG1 followed by separation of the products by thin-layer chromatography revealed that PG1 was an endo-enzyme releasing a mixture of oligosaccharides with tetra-, tri- and digalacturonic acids as the major end-products. Generally the enzyme showed characteristics consistent with many other fungal polygalacturonases, and it macerated and caused cell death in cucumber tissue. The presence of large amounts of PG1 in inoculated potato tubers and its production in vitro by almost all the African Armillaria isolates studied suggests a potential role for the enzyme in the phytopathogenicity of Armillaria .