Department of Cell and Molecular biology, Faculty of Biological Science, Kharazmi University, P.O. Box: 31979-37551, Tehran, Iran.
Enzyme Microb Technol. 2013 Dec 10;53(6-7):406-13. doi: 10.1016/j.enzmictec.2013.09.001. Epub 2013 Sep 12.
Site-directed mutagenesis of an α-amylase isolated from Bacillus megaterium WHO has been performed to evaluate the roles of the calcium binding site residues in enzyme thermostability. The strategy used was to replace residues in the hypothetical calcium binding loops of B. megaterium WHO α-amylase (BMW-amylase) by equivalent positions at Halothermothrix orenii α-amylase (AmyA) as a thermophilic amylase by QuikChange site directed mutagenesis. Asn-75, Ser-76, and His-77 were mutated in the second calcium binding site which resulted in an increase in thermostability. All mutants retained their hydrolytic activity although their kcat parameter decreased in compare to the wild type and in the presence of calcium ions. In S76P and H77E, the Km for starch decreases while overall activity (kcat/Km) was increased. In the presence of calcium, conversion of His-77 to Glu resulted in a 4-fold enhancement in enzyme half life and a 9°C upward shift in T50, which was observed in compare to the wild type. Further analysis suggested the H77E mutant as the most stable which increased the affinity of the enzyme for calcium ion and its optimum temperature was 5°C higher than the wild type.
已对从巨大芽孢杆菌（Bacillus megaterium）中分离得到的α-淀粉酶进行了定点突变，以评估钙结合位点残基在酶热稳定性中的作用。所用的策略是通过易错 PCR 和 QuikChange 定点突变，用嗜热菌海栖热袍菌（Halothermothrix orenii）α-淀粉酶（AmyA）中的等价残基取代巨大芽孢杆菌 WHO α-淀粉酶（BMW-amylase）中假定的钙结合环中的残基，将其转变为热稳定的嗜热淀粉酶。第 2 个钙结合位点中的 Asn-75、Ser-76 和 His-77 发生突变，导致热稳定性增加。所有突变体都保留了水解活性，尽管它们的 kcat 参数与野生型相比有所下降，并且在存在钙离子的情况下也是如此。在 S76P 和 H77E 中，淀粉的 Km 降低，而总活性（kcat/Km）增加。在存在钙的情况下，His-77 突变为 Glu 导致酶半衰期提高了 4 倍，T50 提高了 9°C，与野生型相比有明显的提高。进一步的分析表明，H77E 突变体是最稳定的，它增加了酶对钙离子的亲和力，其最适温度比野生型高 5°C。
