Chen Q M, Liu S H, Ji Y G, Xue Z H, Fu P, Geng H R, Ma M, Sun Q, Liang D, Geng Y Q
College of Life Sciences, Nankai University.
Yi Chuan Xue Bao. 1998;25(3):278-85.
The mutant plasmid pAmy413C, in which G takes the place of A at the 271 position of alpha-amylase gene on the pAmy413 from B. licheniformis, was constructed by site-direct mutagenesis. At the N-terminus of the mature alpha-amylase, amino acid +2Asn was substituted by +3Asp in the wild type protein. Then, the alpha-amylase output of the mutant plasmid pAmy413C in B. subtilis was 2.02-2.57 times higher than that of the wild type pAmy413C in the same strain. The amino acid sequencing at the N-terminus of the matural alpha-amylase revealsed that the recognition site of signal peptidase I moved one amino acid upstream, from Ala-(+2)Asn to AlaAla-(+3) Asp. That is, the +2Asn of the wild type was changed to the +3Asp of the mutant. The secondary structural analysis showed that a 14-cycle structure formed in the alpha-amylase mRNA when the free energy was -51.7 kcal. In this case, the mutant is identical with the wild type. The difference between them is that G at 271 position is no longer paired with U at 211 position, hence, a G-overhang is formed. The secondary structural analysis of protein showed that one amino acid diminished in the turn structure of amino acid at 33-37 position, and this very amino acid is involed in an alpha-helix structure. In short, all the changes mentioned above in conformation and charged amino acids contribute to the increase in the protein secretion in B. subtilis.
通过定点诱变构建了突变体质粒pAmy413C，其中在来自地衣芽孢杆菌的pAmy413上α-淀粉酶基因的第271位，G取代了A。在成熟α-淀粉酶的N端，野生型蛋白质中氨基酸+2Asn被+3Asp取代。然后，突变体质粒pAmy413C在枯草芽孢杆菌中的α-淀粉酶产量比同一菌株中的野生型pAmy413C高2.02 - 2.57倍。成熟α-淀粉酶N端的氨基酸测序表明，信号肽酶I的识别位点向上游移动了一个氨基酸，从Ala-(+2)Asn变为AlaAla-(+3)Asp。也就是说，野生型的+2Asn变为了突变体的+3Asp。二级结构分析表明，当自由能为-51.7千卡时，α-淀粉酶mRNA中形成了一个14环结构。在这种情况下，突变体与野生型相同。它们之间的区别在于，第271位的G不再与第211位的U配对，因此形成了一个G突出端。蛋白质的二级结构分析表明，在第33 - 37位氨基酸的转角结构中减少了一个氨基酸，而这个氨基酸参与了一个α-螺旋结构。简而言之，上述构象和带电荷氨基酸的所有变化都有助于枯草芽孢杆菌中蛋白质分泌的增加。
