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Characterization of the prolyl dipeptidyl peptidase gene (dppIV) from the koji mold Aspergillus oryzae
A Doumas1, P van den Broek, M Affolter
1Nestlé Research Center, 1000 Lausanne 26, Switzerland.
Abstract:
The koji mold Aspergillus oryzae secretes 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 an A. oryzae library by using the A. fumigatus dppIV gene as a probe. Reverse transcriptase PCR experiments showed that the A. oryzae dppIV gene 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 into A. oryzae leads to overexpression of prolyl dipeptidyl peptidase activity, while disruption of the gene abolishes all prolyl dipeptidyl peptidase activity in MMWG. The dppIV null 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 vector Pichia 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.
Insights
Aspergillus oryzae secretes a prolyl dipeptidyl peptidase (DPPIV) that breaks down wheat gluten. This enzyme is not essential for the fungus but may be important for industrial gluten hydrolysis.
Area of Science:
- Microbiology
- Enzymology
- Molecular Biology
Background:
- The koji mold Aspergillus oryzae produces enzymes during fermentation.
- Prolyl dipeptidyl peptidase (DPPIV) activity was observed in A. oryzae grown on wheat gluten.
- The specific role and genetic basis of this DPPIV were not fully understood.
Purpose of the Study:
- To clone and characterize the DPPIV gene from Aspergillus oryzae.
- To investigate the function and necessity of DPPIV in A. oryzae.
- To assess the potential industrial application of A. oryzae DPPIV in gluten hydrolysis.
Main Methods:
- Cloning and sequencing of the A. oryzae dppIV gene using a probe from A. fumigatus.
- Reverse transcriptase PCR to analyze gene structure (exons).
- Gene disruption to create dppIV null mutants and phenotypic analysis.
- Recombinant DPPIV expression in Pichia pastoris for functional complementation.
Main Results:
- The A. oryzae dppIV gene was cloned and sequenced, revealing a two-exon structure.
- The gene encodes a precursor polypeptide secreted as a 95-kDa glycoprotein.
- Disruption of the dppIV gene eliminated DPPIV activity without altering fungal phenotype.
- Loss of DPPIV activity affected peptide profiles in culture broth, which was reversed by recombinant DPPIV.
Conclusions:
- A. oryzae DPPIV is a secreted glycoprotein encoded by a specific gene.
- DPPIV activity is not essential for A. oryzae's growth or phenotype under the tested conditions.
- A. oryzae DPPIV shows potential for industrial applications in hydrolyzing proline-rich substrates like wheat gluten.