A simple fractionation protocol for, and a comprehensive study of the molecular properties of, two major endopolygalacturonases from Aspergillus niger.

A comprehensive study on purification and characterization of the two endopolygalacturonases from Aspergillus niger, PG II and PG IV, accounting for 70% of the total polygalacturonase activity, is reported. These enzymes were purified to homogeneity using ion-exchange chromatography and gel filtration. The enzymes had specific activities of 982 and 3750 units/mg, and their molecular masses were 61 and 38 kDa, respectively. The pH optimum of PG II was pH 3.8-4.3 and for PG IV it was between pH 3 and 4.6, and the temperature optima also differed for the enzymes. The enzymes preferred pectic acid as a substrate, cleaving it at random, leading to the release of oligogalacturonides as products. The K(m) values of the two enzymes were found to be 0.12 and 0.72% respectively. The enzymes were rich in hydrophilic amino acids and relatively low in the sulphur-containing amino acids. Both enzymes were rich in beta-structure and differed in their tertiary folding. The tryptophan residues were in a hydrophobic environment. The enzymes differed in their thermal stability; the midpoint of thermal inactivation, T(m), of the two enzymes was found to be 43 degrees C for PG II and 46 degrees C for PG IV.