Purification, characterization and mode of effect of another endo-D-galacturonanase from Aspergillus niger

Abstract

A mixture of pectolytic enzymes was isolated from a commercial preparation of Pectinex Ultra. endo-D-Galacturonanase (EC 3.2.1.15) was obtained from this mixture of enzymes, produced by Aspergillus niger cultures, by affinity chromatography using cross-linked pectic acid as a support and by subsequent chromatography on a Sephadex G-100 column. One form of endo-D-galacturonanase only of approximate molecular weight of 35 000 was detected in the purified product by electrophoresis in polyacrylamide gel. The enzyme showed maximal activity and stability at pH 4.9 and 40°. The mode of degradation of a high molecular weight substrate and the per cent of the glycosidic bonds cleaved (6%) at a viscosity decrease by 50% indicate an endo type action pattern. endo-D-Galacturonanase was characterized by the mode of cleavage and the kinetic constants Km and V for oligogalacturonic acids and polygalacturonic acid. On the basis of the knowledge of the kinetic constants and the degradation products the mode of action of the enzyme and the extent of its active site are discussed.