UTILIZATION OF THE TEF1-ALPHA GENE [TEF1) PROMOTER FOR EXPRESSION OF POLYGALACTURONASE GENES, PGAA AND PGAB, IN ASPERGILLUS-ORYZAE

For the development of an efficient gene expression system in a shoyu koji mold Aspergillus oryzae KBN616, the TEF1 gene, encoding translati on-elongation factor la, was cloned from the same strain and used for expression of polygalacturonase genes. The TEF1 gene comprised 1647 bp with three introns. The TEF1-alpha protein consisted of 460 amino aci ds possessing high identity to other fungal TEF proteins. Two nucleoti de sequences homologous to the upstream activation sequence, character ized for the ribosomal protein genes in Saccharomyces cerevisiae, as w ell as the pyrimidine-rich sequences were present in the TEF1 gene pro moter region, suggesting that the A. oryzae TEF1 gene has a strong pro moter activity. Two expression vectors, pTFGA300 and pTFGB200 for prod uction of polygalacturonases A and B respectively, were constructed by using the TEF1 gene promoter. A polygalacturonase (PGB) gene cloned f rom the same strain comprised 1226 bp with two introns and encoded a p rotein of 367 amino acids with high similarity to other fungal polygal acturonases. PGA and PGB were secreted at approximately 100 mg/l in gl ucose medium and purified to homogeneity. PGA had a molecular mass of 41 kDa, a pH optimum of 5.0 and temperature optimum of 45 degrees C. P GB had a molecular mass of 39 kDa, a pH optimum of 5.0 and temperature optimum of 55 degrees C.