In a molecular screening program to select a potent thermostable amylase from a previously isolated thermophiles, a locally isolated, thermophilic lipase-producing Geobacillus thermoleovorans YN (accession number AF385083), was shown to secrete a thermostable α-amylase constitutively. The optimal enzyme activity was measured at 75°C, where 90% of the activity was retained at 80°C after one hour of incubation. A catabolite repression due to the addition of glucose to the basal salt medium was demonstrated, while 4 folds increase in volumetric production was achieved in LB and starch-supplemented basal salt media and presented in SDS-PAGE and zymogram. A blunt end PCR fragment (2146 bp) was amplified from genomic DNA using a designed set of primers and ligated to Bluescript —II KS(+) vector, transformed to E. coli DH5-α competent cells by electroporation and screened on LB-agar plates induced with IPTG. Nucleotide sequencing of selected clone revealed two ORFs, the first was (GTG) with a molecular size 1649 nucleotides encoding 549aa residues of a predicted molecular weight 62.592 kD and the second (ATG) with a molecular size 1613 nucleotides encoding 537aa residues of a predicted molecular weight 61.04 kD.
