Multiple amylase genes in two strains of Bacillus stearothermophilus

Plasmid DNA fragments from Bacillus stearothermophilus ATCC29609 (BR135) and chromosomal DNA fragments from B. stearothermophilus ATCC31195 (BR132) were cloned into pBR322 and transformed into Escherichia coli strain HB101. Clones were selected which demonstrate extracellular expression of thermostable amylase. The DNA inserts from both strains showed similar restriction maps. Examination of the parental strains revealed plasmids of approx. 100 kb and 35 kb for BR135 and no discernible plasmids for BR132. Hybridization experiments showed that the amylase gene is contained within a 3.2-kb HindIII fragment on both plasmids, and to be present in multiple copies in the BR135 chromosome. Multiple chromosomal copies of the amylase gene were also observed for BR132. Purification of the alpha-amylase produced by the cloned plasmid amy gene yielded a homogeneous 58-kDa protein with thermostable amylase activity.