Purification and characterization of an endopolygalacturonase from Verticillium albo-atrum

A polygalacturonase was isolated from the culture filtrate of the fungal plant pathogen Verticillium albo-atrum and purified 22-fold to homogeneity as judged by SDS-electrophoresis. The enzyme was a basic protein with a molecular weight of 37 kDa, an isoelectric point greater than or equal to 8.6 and containing 1.7% carbohydrate. The enzyme was an endo-polygalacturonase and hydrolysed a wide range of pectic substrates including polygalacturonic acid, 93% methylated pectin and pectins in tomato cell walls. The best substrate was 31% methylated pectin. Relative reaction rates on pectins with different degrees of methylation could be explained by considering both the number of susceptible bonds and non-specific enzyme-substrate interactions. The principal products of long-term hydrolysis were di- and mono-galacturonate. Maximum activity was observed at pH 4.6-5.0 and 46 degrees C. However, the enzyme lost activity above 30 degrees C in the absence of substrate. Enzyme activity was very sensitive to changes in ionic strength at low salt levels. It was stable in the pH range 3-11 at 30 degrees C.