Nucleotide sequence and expression of the isoamylase gene from an isoamylase-hyperproducing mutant, Pseudomonas...

The isoamylase gene (ISO) of Pseudomonas amyloderamosa JD210, an isoamylase-hyperproducing mutant, was cloned in an isoamylase-deficient and transformable mutant strain K31. By deletion analysis, the ISO gene was found to be located within a 3.3 kilobases BamHI fragment. Its nucleotide sequence contained an open reading frame of 2328 nucleotides (776 amino acids) encoding a secreted isoamylase precursor. The ISO gene fragment was inserted into plasmids pKT230 and pBR 322 in opposite orientations. The expression of the ISO gene in the constructed plasmids was compared in P. amyloderamosa K31, Pseudomonas aeruginosa PAO1-161, Pseudomonas putida mt-2 and Escherichia coli HB101. In all transformed cells, the majority of the isoamylase produced was secreted and higher isoamylase activities were obtained in transformats with the transcriptional direction of the ISO gene similar to the nearby drug-determinant gene of the vector.