State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
J Agric Food Chem. 2023 Apr 26;71(16):6480-6489. doi: 10.1021/acs.jafc.3c00665. Epub 2023 Mar 23.
The efficient production of high-value-added bioproducts from starchy substances requires α-amylases with hyperthermophilic properties for industrial starch liquefaction. In this study, two hyperthermophilic α-amylases with significant differences in thermostability, Amy and Amy, were comparatively studied through structural analysis, domain swapping, and site-directed mutagenesis, finding that three residues, His152, Cys166, and His168, located in domain B were the main contributors to hyperthermostability. The effects of these three residues were strongly synergistic, causing the optimum temperature for the mutant K152H/A166C/E168H of Amy to shift to 95-100 °C and stabilize at 90 °C without Ca. Compared to Amy and Amy, the mutant K152H/A166C/E168H, respectively, exhibited 1.7- and 2.5-times higher starch hydrolysis activity at 105 °C and pH 5.5 (10411 ± 70 U/mg) and released 1.1- and 1.7-times more maltooligosaccharides from 1% starch. This work has interpreted the hyperthermophilic mechanism of α-amylase and thereby providing a potential candidate for the efficient industrial conversion of starch to bioproducts.
从淀粉质物质高效生产高附加值的生物制品需要具有嗜热特性的α-淀粉酶，用于工业淀粉液化。在这项研究中，通过结构分析、结构域交换和定点突变，比较研究了两种热稳定性差异显著的嗜热α-淀粉酶 Amy 和 Amy，发现位于结构域 B 中的三个残基 His152、Cys166 和 His168 是导致超嗜热性的主要原因。这三个残基的影响具有很强的协同作用，导致 Amy 的突变体 K152H/A166C/E168H 的最适温度转移到 95-100°C，并在没有 Ca2+的情况下在 90°C 稳定。与 Amy 和 Amy 相比，突变体 K152H/A166C/E168H 在 105°C 和 pH5.5 时的淀粉水解活性分别提高了 1.7 倍和 2.5 倍（10411±70 U/mg），从 1%淀粉中释放的麦芽寡糖分别增加了 1.1 倍和 1.7 倍。这项工作解释了α-淀粉酶的嗜热机制，从而为高效工业转化淀粉为生物制品提供了一个潜在的候选酶。
