Stabilisation in bacillus stearothermophilus of a recombinant plasmid carrying the homologous α‐amylase gene

The phenotypic stability of the recombinant plasmid pAT9 (11.5 MD), which contained the cloned α‐amylase gene from B. stearothermophilus, was studied in batch and continuous culture. Irrespective of the type of culture (batch or continuous; or using glucose or maltose as carbon source), deletion plasmids of the phenotype KmrAmy− the same size (7.3 MD) emerged with time. DNA sequencing analysis and Southern hybridization of a region adjacent to both ends of the pAT9 HindIII fragment containing the α‐amylase gene showed that almost the entire part of the HindIII fragment was lost. Homology between fragments contiguous to the pAT9 HindIII terminals allowed exclusion of a fragment that did not encode the α‐amylase gene and a new recombinant plasmid, pATHP9 (KmrAmy+; 7.5 MD), was constructed which had enhanced phenotypic stability in B. stearothermophilus.