The exo-polygalacturonase from Aspergillus tubingensis: characterization and cloning of the gene

Abstract

Abstract From the culture fluid of the hyphal fungus Aspergillus tubingensis an exopolygalacturonase (PGX) was isolated and purified. Besides the physico-chemical properties of the purified enzyme, the mode of action and the kinetics were studied on polymeric and oligomeric substrates. The enzyme hydrolyses galacturonic acid from the non-reducing end of polygalacturonic acid and from oligogalacturonates with different degree of polymerization (DP=2–7). Kinetic analysis data on non-reduced and reduced oligogalacturonates were used for the calculation of the subsite affinities to obtain more insight in the substrate binding and catalysis on the molecular level. The PGX encoding gene, pgaX, was cloned by reverse genetics and the nucleotide sequence was determined. Restricted amino acid sequence identity is found with other polygalacturonases and a phylogenetic tree has been constructed with fungal, bacterial and plant polygalacturonases. The expression of PGX is induced by galacturonic acid and repressed by glucose at the level of transcription.