Sixty-nine local bacterial isolates of thermophilic Bacillus spp. isolated from different soil locations over Iraq land were screened for their ability to produce extracellular α-amylase (EC 3.2.1.1) in solid and submerged cultures. The isolate Bacillus stearothermophilus M13 was selected based on its production of enzyme among the other isolates and used in the present work to study the production,purification and chracterization of enzyme then the cloning of the α-amylase gene. It was susceptible for some antibiotics (Imipenem, Cefotaxime, Norfloxacin, Gentamicin, Pencillin, Ampicillin). while, it was resistant to Aztreonem.The optimum conditions for α-Amylase production from Bacillus stearothermophilus M13 were grown in TSM medium with an initial pH 7.0 at 55oC for 24 hrs of incubation, the activity of crude extract was 0.04 U/ml. α-Amylase was purified from Bacillus stearothermophilus M13 isolate by several steps including saturation of crude extract with ammonium sulphate (40-80%), ion exchange chromatography by DEAE-Sephadex in which four isozymes(forms) were noticed named as (a,b.c and d). “b form”, which showed the higher specific activity among the others, was applied to gel filtration through Sephadex-G100 that revealed overall specific activity of 1.675 U/mg ; 30.45 folds of purification and yield of 50.25 %. The characterization of the partial purified enzyme showed that its molecular weight was “56234” dalton as determined by gel filtration technique, on the other hand the estimation of molecular weight using SDS- PAGE was “55426” dalton.The enzyme activity was higher at pH rang 6.5-7.5 and it showed stability at pH rang 6.5-8.0. The maximum enzyme activity was reported at 60oC and it was stable at 70oC for 30 min and retained 50% of its original activity at 90ºC, which confirmed that this enzyme is heat stable enzyme.The α-amylase showed antigenic activity in vivo ( Rabbits) ,the polyclonal antibodies could decrease the α-amylase activity with 37.4% . Then the products of α-amylase action on the starch over the reaction time had been investigated using TLC , glucose ,maltose and other unknown carbohydrates were produced that could not be identified due to unvaiability of standard analoges .Immobilization of α-amylase via entrabment method using the calcium alginate was successful, it retained 24% of its original activity on the 25th day of storage when it kept at 4ºC.α-Amylase genome was investigated for any possible plasmid DNA carrying the α-amylase gene , the plasmid DNA was isolated by three different protocols, ( Alkaline lyses ;CTAP and Mielenz`s protocol ) results showed no detectable plasmid.Cloning of α-amylase gene was done ; chromosomal DNA of B. stearothermophilus M13was extracted and partially digested with Hind III . The pBR322 was isolated and purified via CsCl-EtBr gradient ultracentrifugation, then it completely digested by HindIII. The digested chromosomal DNA and vector was ligated by T4 ligase in ratio of the inserted DNA : vector DNA as 5:1 , followed by the transformation of the host cells ( E. coli ) with hybrid vector. Transformants (3847) were selected according to the ampicillin resistance as in the primary screening then to hydrolysis of starch. Five clones ( 0.13 %) only out of 3847 transforments were able to produce α-amylase on solid and liquid media , the higher transformant-producer was named E. coli MA1 , in which the specific activity was 0.045 U/mg .
