Characterization of the Prolyl Dipeptidyl Peptidase Gene (dppIV) from the Koji Mold Aspergillus oryzae

Abstract

ABSTRACTThe koji moldAspergillus oryzaesecretes a prolyl dipeptidyl peptidase (DPPIV) when the fungus is cultivated in a medium containing wheat gluten as the sole nitrogen and carbon source (MMWG). We cloned and sequenced the DPPIV gene from anA. oryzaelibrary by using theA. fumigatus dppIVgene as a probe. Reverse transcriptase PCR experiments showed that theA. oryzae dppIVgene consists of two exons, the first of which is only 6 bp long. The gene encodes an 87.2-kDa polypeptide chain which is secreted into the medium as a 95-kDa glycoprotein. Introduction of this gene intoA. oryzaeleads to overexpression of prolyl dipeptidyl peptidase activity, while disruption of the gene abolishes all prolyl dipeptidyl peptidase activity in MMWG. ThedppIVnull mutants did not exhibit any change in phenotype other than the absence of prolyl dipeptidyl peptidase activity, suggesting that this activity is not essential. This loss of activity diminished the number of dipeptides and increased the number of larger peptides present in the MMWG culture broth. These effects were reversed by the addition of purified, recombinant DPPIV from the methylotrophic yeast expression vectorPichia pastoris. Our results suggest that the DPPIV enzyme may be of importance in industrial hydrolysis of what gluten-based substrates, which are rich in Pro residues.