Purification, Characterization and Amino Acid Sequence of Endopolygalacturonases IVa and IVb from Fungus Stereum purpureum

Abstract

Two endopolygalacturonases (EndoPG IVa and IVb) were purified from the culture filtrate of Stereum purpureum (Chondrostereum purpureum) , the causative fungus of apple silver-leaf disease, by three steps of column chromatography. Respective molecular masses were determined at 36,017 and 37,266 Da using ESI-MS. Deglycosylation with endo-β-N-acetylglucosaminidase reduced molecular masses to 34,987 and 35,202 Da, reductions corresponding to the loss of one and two high-mannose type M5 sugar chains, respectively. From this result, EndoPG IVa and IVb are assumed to share a common primary structure. Their thermal stabilities were up to 55℃, considerably lower than that of EndoPG Ia (up to 70℃). Melting temperature (T_m) values for EndoPG IVb and EndoPG Ia were 62 and 79.5℃ respectively. Specific activities and K_m values measured for EndoPG IVa and IVb were almost identical to those of EndoPG Is. Amino acid sequences were deduced by 3′ and 5′ RACEs cloning of cDNA based on both EndoPG IVs sharing an identical N-terminus (Accession No. AB252456). No large deletion of C-terminal amino acid residues (44 residues) was observed in EndoPG IV, as has been reported for mature EndoPG I. Amino acid sequence homology of EndoPG IV with EndoPG I was found to be 72%.