Kim C H, Sata H, Taniguchi H, Maruyama Y
Department of Agricultural Chemistry, University of Tokyo, Japan.
Biochim Biophys Acta. 1990 Apr 6;1048(2-3):223-30. doi: 10.1016/0167-4781(90)90060-f.
The raw potato-starch-digesting alpha-amylase gene of Bacillus circulans F-2 was cloned for the first time in Escherichia coli C600, using plasmid pYEJ001. The recombinant plasmid, named pYKA3, has a 5.4 kb insert from a chromosome of the donor bacterium. Subcloning of this amylase gene gave plasmid pHA300 which carried 3.15 kb of the inserted DNA. The transformed bacterium, E. coli C600 (pYKA3), produced the amylase in the periplasmic space, whereas it is secreted outside the cell in the donor bacterium. The cloned raw-starch-digesting alpha-amylase has a molecular weight of 93,000 on SDS-PAGE, and its action pattern was absolutely the same as that of the potent raw-starch-digestible amylase produced by B. circulans F-2. The periplasmic amylase produced by the transformed E. coli (pHA300) could digest raw starch granules such as potato, corn and barley raw starch granules, indicating that the raw-starch-digesting amylase is active in E. coli. Furthermore, this amylase crossreacted with the rabbit antiserum raised against the raw potato-digesting alpha-amylase of B. circulans F-2. From these results it was concluded that the cloned amylase is the same amylase protein as B. circulans F-2 amylase, which has a potent raw-starch digestibility. Thus, this paper is to our knowledge the first describing the molecular cloning of raw-starch-digesting alpha-amylase from Bacillus species and its successful expression in E. coli.
利用质粒pYEJ001，首次在大肠杆菌C600中克隆了环状芽孢杆菌F-2的生马铃薯淀粉消化性α-淀粉酶基因。重组质粒命名为pYKA3，其插入片段来自供体细菌染色体，长度为5.4 kb。该淀粉酶基因的亚克隆得到了携带3.15 kb插入DNA的质粒pHA300。转化菌大肠杆菌C600 (pYKA3)在周质空间产生淀粉酶，而在供体细菌中淀粉酶分泌到细胞外。在SDS-PAGE上，克隆的生淀粉消化性α-淀粉酶分子量为93,000，其作用模式与环状芽孢杆菌F-2产生的高效生淀粉消化性淀粉酶完全相同。转化后的大肠杆菌(pHA300)产生的周质淀粉酶能够消化生淀粉颗粒，如马铃薯、玉米和大麦生淀粉颗粒，这表明生淀粉消化性淀粉酶在大肠杆菌中具有活性。此外，这种淀粉酶与针对环状芽孢杆菌F-2的生马铃薯消化性α-淀粉酶产生的兔抗血清发生交叉反应。从这些结果可以得出结论，克隆的淀粉酶与环状芽孢杆菌F-2淀粉酶是相同的淀粉酶蛋白，具有很强的生淀粉消化能力。因此，据我们所知，本文首次描述了从芽孢杆菌属中克隆生淀粉消化性α-淀粉酶及其在大肠杆菌中的成功表达。
