Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin, China.
J Appl Microbiol. 2012 Sep;113(3):541-9. doi: 10.1111/j.1365-2672.2012.05359.x. Epub 2012 Jul 10.
The purpose of this research was to obtain the mutant of Bacillus licheniformis alpha amylase (BLA) with an improved acid stability and elucidate the difference in catalytic mechanism under acidic conditions between wild-type and mutant BLAs.
The stability of BLA under acid condition was enhanced through direct evolution using error-prone polymerase chain reaction. Two mutation sites, T353I and H400R, were obtained in BLA. To identify the mutation of amino acids in Thr353Ile/His400Arg related to its acid stability, single mutants Thr353Ile and His400Arg were obtained via site-directed mutagenesis. Among the resulting mutant enzymes, the k(cat)/K(m) values of the mutants Thr353Ile, His400Arg and Thr353Ile/His400Arg under pH 4.5 were 3.5-, 6.0- and 11.3-fold higher, respectively, than that of the wild-type. Thr353Ile/His400Arg exhibited stronger tolerance towards a lower pH without obvious difference in thermostability when compared with wild-type.
The results combined with three-dimensional structure analysis of mutant BLAs demonstrated that Thr353Ile/His400Arg showed an improved acid stability under low pH condition as a result of the interactions of hydrogen bonding, hydrophobicity, helix propensity and electrostatic fields.
It provides theoretical basis and background data for the improvement of acid stability in BLA by protein engineering.
本研究旨在获得具有改良酸性稳定性的地衣芽孢杆菌α-淀粉酶（BLA）突变体，并阐明野生型和突变型 BLAs 在酸性条件下催化机制的差异。
通过易错聚合酶链反应直接进化提高了 BLA 在酸性条件下的稳定性。在 BLA 中获得了两个突变位点，T353I 和 H400R。为了确定 Thr353Ile/His400Arg 氨基酸突变与其酸性稳定性的关系，通过定点突变获得了单个突变体 Thr353Ile 和 His400Arg。在所得突变酶中，突变体 Thr353Ile、His400Arg 和 Thr353Ile/His400Arg 在 pH4.5 下的 k(cat)/K(m) 值分别比野生型高 3.5、6.0 和 11.3 倍。与野生型相比，Thr353Ile/His400Arg 在较低 pH 值下表现出更强的耐受性，而热稳定性没有明显差异。
结果结合突变 BLAs 的三维结构分析表明，Thr353Ile/His400Arg 在低 pH 条件下表现出改善的酸性稳定性，这是由于氢键、疏水性、螺旋倾向和静电场相互作用的结果。
它为通过蛋白质工程提高 BLA 的酸性稳定性提供了理论依据和背景数据。
