Ji Y, Yang L, Chen Q, Ma M, Geng Y, Jiang R
Lab. for Biotechnology, Nankai Univ., Tianjin.
Yi Chuan Xue Bao. 1994;21(3):227-34.
By site-directed mutagenesis, G and C have taken the place of T and G at nucleotide sequence 287 and 291 of B. licheniformis alpha-amylase gene to generate pAm-y413B and the N-terminal sequence of mature protein have been changed from 7Leu 8Met to 7Arg8Ile. By the insertion of polylinker into the C-terminal of the signal sequence of alpha-amylase gene of pAmy413, the signal peptide of alpha-amylase produced by pAmy413L is 13 amino acids more than the pAmy413 (which is 29 amino acids long) and also, a new recognition cleavage sequence for signal peptidase I (Ala-Gln-Ala decreases Ser) is created; The secondary structure of the signal peptide has been analyzed by computer programs. The alpha-amylase relative activity of the two mutant strains is 3% and 36% of pAmy413, respectively. The molecular weight of extracellular alpha-amylase is the same as pAmy413. Terminal analysis shows that the N-terminal amino acid of mature protein is Ala, not Ser, and suggests that SPase I prefers to cleavage at the wild type recognition site (Ala-Ala-Ala decreases Ala). Therefore, all of the above results show that the secretion of alpha-amylase in B. subtilis is in accordance with the co-translational transportation model.
通过定点诱变，在地衣芽孢杆菌α-淀粉酶基因的核苷酸序列287和291处，G和C取代了T和G，从而产生了pAm-y413B，成熟蛋白的N端序列从7Leu 8Met变为7Arg8Ile。通过将多克隆位点插入pAmy413的α-淀粉酶基因信号序列的C端，pAmy413L产生的α-淀粉酶信号肽比pAmy413（其长度为29个氨基酸）多13个氨基酸，并且还产生了信号肽酶I的新识别切割序列（Ala-Gln-Ala减少Ser）；信号肽的二级结构已通过计算机程序进行分析。两种突变菌株的α-淀粉酶相对活性分别为pAmy413的3%和36%。胞外α-淀粉酶的分子量与pAmy413相同。末端分析表明，成熟蛋白的N端氨基酸是Ala，而不是Ser，这表明信号肽酶I更倾向于在野生型识别位点（Ala-Ala-Ala减少Ala）处切割。因此，上述所有结果表明，枯草芽孢杆菌中α-淀粉酶的分泌符合共翻译转运模型。
