Purification and characterization of two inducible exopolygalacturonases from Aspergillus kawachii

Abstract

Of two exopolygalacturonases purified and characterized from an Aspergillus kawachii culture grown on lemon pomace, the main one, exoPG1, was a glycosylated protein with a molecular mass of 75 kDa, isoelectric point in the 4.00–4.65 pH range, and a 3.0–4.0 pH optimum, though with activity at pH 2.0. ExoPG1 cleaved monomer units irrespective of the degree of substrate polymerization. Di- and trigalacturonic acids were completely hydrolyzed, whereas polygalacturonic acid (PGA) only incompletely. ExoPG1, along with a recombinant endoPG from the same fungal strain, was necessary for the hydrolysis of PGA down to the monomer. pH stability was maximum in the range 4.0–5.0 irrespective of the incubation temperature and decreased as the temperature increased from 30 to 70 °C. The enzyme appeared not to require divalent cations for activity. Protein identification by MALDI-TOF-TOF MS/MS indicated homology of exoPG1 with the exopolygalacturonase PGXB of Aspergillus. niger, an exopolygalacturonase of Aspergillus tubingensis, and the exopolygalacturonase X of Aspergillus kawachii, a hypothetical enzyme predicted from the complete sequencing of the genome of the fungus. Both these latter proteins are unusual in that they have identical primary sequences. We therefore conclude that exoPG1 is probably the hypothetical A. kawachii exopolygalacturonase X. ExoPG2—having a molecular weight of 80 kDa, an isoelectricpoint between pHs 4.5 and 5.0, a 4.0 pH optimum, and kinetics with PGA similar to those of exoPG1—shared similarities with the exopolygalacturonase PGXC of A. niger and another proposed exopolygalacturonase of A. kawachii. This report is the first concerning exopolygalacturonases from A. kawachii.