CLONING AND SEQUENCE-ANALYSIS OF CDNA-ENCODING ENDOPOLYGALACTURONASE-I FROM STEREUM-PURPUREUM

Endopolygalacturonase (endoPG) I was obtained from Stereum purpureum b y an improved easier purification procedure. It was found that EndoPG I consisted of three glycosilated proteins with the same isoelectric p oint and different molecular masses, 42, 45, and 48 kDa, respectively. However, the enzymatic deglycosilation product of endoPG I gave a sin gle band at the position corresponding to 39 kDa on SDS-PAGE. Furtherm ore, the N-terminal amino acid sequences of three endoPGs were identic al one another up to 20 residues. A cDNA library was constructed and p ositive cDNA clones encoding endoPG I were isolated by using antibody raised against the purified endoPG I. Nucleotide sequence analysis of the cDNA disclosed a 1212-bp open reading frame that encoded 403 amino acid residues. The N-terminal amino acid sequence (residues 1-20) of endoPG I coincided with the deduced amino acid sequence starting from the 25th residue. Therefore, the sequence of the first 24 residues rep resented a signal peptide and the remaining sequence, consisting of 37 9 residues, was the mature protein with molecular mass of 39.1 kDa. Th e deduced sequence of endoPG I showed 30-45% similarity in comparison with those of bacterial and fungal endoPGs, and the sequence of putati ve active site residues reported for the endoPGs was highly conserved in the sequence of endoPG I.