Medium Temperature Alpha-amylase From Bacillus Subtilis Gene Cloning And Expression In Prokaryotic Microorganisms

Alpha-amylase(1,4-alpha-D-glucan-glucanohydrolase,EC3.2.1.1)hydrolyzes alpha-1,4-D-linkages in starch. The action of alpha-amylase on starch leads to a rapid decrease of starch solution viscosity. Alpha-amylase is widely applied in numerous areas, such as starch saccharificaton, fermentation, spin, paper. Thus any researches on alpha-amylase are particular significant in both industorial production and scientific investigation.A bacterium B-10 with the capability of degrading starch, identified as Bacillus subtilis, is isolated from the soil located in Mountain Huo Yan of Xin Jiang province. The alpha-amylase (AMY-1) from B-10 has been purified and characterized. AMY-1 has optimum activity for the reaction at 70℃and pH 5.5 with soluble starch as a substrate, also shows good thermal and pH stability compared with the alpha-amylase (AMY-2) from the market. Most metal ions and chemicals have no effect on the activity of AMY-1.The alpha-amylase gene is obstained by PCR using specific primers designed according to the known other alpha-amylase nucleotide sequences in Bacillus sp. The alpha-amylase gene is 1980bp long, comprising one open reading frame encoding a polypeptide of 660 amino acids which includes a signal peptide of 32 amino acids. The nucleotide sequence of alpha-amylase gene shows the homologie with those alpha-amylase genes deposited in GenBank, one of which exhibits highest homology of 92%. The alpha-amylase gene is inserted into the vector pET-30a, and expression products bore normal bioactivity in E. coli.The promoter of alpha-amylase gene of B-10 is cloned, then the alpha-amylase gene with the promoter and signal sequences is inserted into the vector pUBC-19, and the expression product bore normal bioactivity in Bacillus subtilis WB800, too. At the same time the alpha-amylase expressed in Bacillus subtilis WB800 is 28 times higher than that in the B-10.