CLONING AND NUCLEOTIDE-SEQUENCE OF THE ACID PROTEASE-ENCODING GENE (PEPA) FROM ASPERGILLUS-ORYZAE

We have cloned a genomic DNA sequence encoding the acid protease (PEPA ) from Aspergillus oryzae using a 0.6-kb fragment as a probe. This fra gment was amplified by the polymerase chain reaction (PCR) using oligo nucleotide primers designed from the partial amino acid sequences of p eptide fragments of the purified protein. Nucleotide sequencing analys is has shown that the cloned gene (designated pepA) encodes 404 amino acid residues and contains 3 putative introns ranging in length from 5 0 to 61 nucleotides. The deduced amino acid sequence of the A. oryzae PEPA has a high degree of homology (67%) to the A. awamori PEPA. Compa rison with the amino acid sequence of A. awamori PEPA suggests that th e A. oryzae PEPA may consist of a 78 amino acid prepro-peptide and 326 amino acid mature protein. The amino acid composition of the mature p rotein was almost consistent with that of the acid protease purified f rom A. oryzae reported previosuly. Southern hybridization analyses sho wed that the pepA gene exists as a single copy in the A. oryzae chromo some. The cloned gene was found to be functional, since transformants of A. oryzae containing multiple copies of the pepA gene showed a 2-6 fold increase in acid protease activity compared with the recipient st rain.