Sohma A, Fujita T, Yamane K
Institute of Biological Sciences, University of Tsukuba, Ibaraki, Japan.
J Gen Microbiol. 1987 Nov;133(11):3271-7. doi: 10.1099/00221287-133-11-3271.
A thermostable alpha-amylase gene (amyT631) from Bacillus stearothermophilus A631 was cloned into pBR322 and recloned into pUB110: the resulting plasmid was designated pTUB607. To investigate the processing from preproenzyme to mature enzyme, amyT631 from pTUB607, after digestion with BAL31, was introduced into the B. subtilis alpha-amylase secretion vector pTUB285. Three chimaeric plasmids, pTUB613, pTUB616, and pTUB617, were isolated. The fused alpha-amylases expressed from the three plasmids seemed to be synthesized as preproenzymes. From analysis of the NH2-terminal amino acid sequences of purified extracellular alpha-amylases, the precursors of the fused enzymes appeared to be cleaved at first between amino acids 31 and 32 from the translation initiator Met (positions -11 and -10 with respect to the beginning of the mature enzyme), and processed to mature extracellular enzymes in which the NH2-terminal amino acid sequences were the same as that of the parental pTUB607 alpha-amylase, in spite of the lengths of the prosequences and the amino acid composition near the secondary cleavage sites being different in each enzyme.
将嗜热脂肪芽孢杆菌A631的一个热稳定α-淀粉酶基因（amyT631）克隆到pBR322中，然后再克隆到pUB110中：所得质粒命名为pTUB607。为了研究从前体酶到成熟酶的加工过程，用BAL31消化后，将pTUB607中的amyT631导入枯草芽孢杆菌α-淀粉酶分泌载体pTUB285。分离得到了三个嵌合质粒，pTUB613、pTUB616和pTUB617。从这三个质粒表达的融合α-淀粉酶似乎以前体酶的形式合成。通过对纯化的细胞外α-淀粉酶的NH2末端氨基酸序列分析，融合酶的前体似乎首先在翻译起始Met的第31和32个氨基酸之间（相对于成熟酶起始位置为-11和-10位）被切割，并加工成细胞外成熟酶，尽管每种酶的前导序列长度和二级切割位点附近的氨基酸组成不同，但其NH2末端氨基酸序列与亲本pTUB607α-淀粉酶的相同。
