:Cloning of α-Amylase Structural Gene and Analysis of α-Amylase Production Using the Cloned Gene

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The α-amylase structural gene (ainyE+) maps near the arol locus on the Bacillus subtilis chromosome. The gene order around amyE+ is lin2-tmrA-amyR-amyE-tmrB-arol. A DNA fragment containing amyR2, amyE+, arol+ was cloned in 13. subtilis temperate phage P11 by the prophage transformation method. Partial diploids in the amyE+ gene were constructed by the transfer of another B. subtilis α-amylase gene, which showed different electrophoretic mobility in 7.5% polyacrylamide gel, into the chromosome of a strain lysogenic for the isolated specialized transducing phage (ρlld amyR2 afnyE+ arol+). The tmrA and amyR2 genes, which are regulatory genes forr the expression of amyE+, acted as cis-dominant on the hyperproduction of the enzyme. plld amyR2 amyE+ trrB arol+ DNA was partially digested by a restriction enzyme Sau 3A and was ligated with a plamid pUB 110 by T4 DNA ligase after pUB 110 DNA was cleaved by Barn-HI. The constructed plasmid, pTUB 4, have the 2.3 kilobase pairs insert containing the annyR 2 and amyE+ genes, α-Amylase expressed from pTUB4 was secreted into the culture medium and the enzyme activity secreted was neutralized by a rabbit anti-serum against B. subtilis α-amylase.
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