多点突变提高解淀粉芽孢杆菌α-淀粉酶的热稳定性。

Improving thermostability of Bacillus amyloliquefaciens alpha-amylase by multipoint mutations.

机构信息

National Engineering Laboratory for High-efficient Enzyme Expression, Fuzhou, Fujian, China; The Key Laboratory of Marine Enzyme Engineering of Fujian Province, Fuzhou University, Fuzhou, Fujian, China; College of Biological Science and Engineering, Fuzhou University, Fuzhou, Fujian, China.

出版信息

Biochem Biophys Res Commun. 2023 Apr 23;653:69-75. doi: 10.1016/j.bbrc.2023.02.064. Epub 2023 Feb 24.

DOI:10.1016/j.bbrc.2023.02.064

PMID:36857902

Abstract

The medium-temperature alpha-amylase of Bacillus amyloliquefaciens is widely used in the food and washing process. Enhancing the thermostability of alpha-amylases and investigating the mechanism of stability are important for enzyme industry development. The optimal temperature and pH of the wild-type BAA and mutant MuBAA (D28E/V118A/S187D/K370 N) were all 60 °C and 6.0, respectively. The mutant MuBAA showed better thermostability at 50 °C and 60 °C, with a specific activity of 206.61 U/mg, which was 99.1% greater than that of the wild-type. By analyzing predicted structures, the improving thermostability of the mutant MuBAA was mainly related to enhanced stabilization of a loop region in domain B via more calcium-binding sites and intramolecular interactions around Asp187. Furthermore, additional intramolecular interactions around sites 28 and 370 in domain A were also beneficial for improving thermostability. Additionally, the decrease of steric hindrance at the active cavity increased the specific activity of the mutant MuBAA. Improving the thermostability of BAA has theoretical reference values for the modification of alpha-amylases.

摘要

地衣芽孢杆菌中温 α-淀粉酶广泛应用于食品和洗涤加工过程。提高 α-淀粉酶的热稳定性并研究其稳定性机制对于酶工业的发展非常重要。野生型 BAA 和突变体 MuBAA（D28E/V118A/S187D/K370N）的最适温度和 pH 均为 60°C 和 6.0。突变体 MuBAA 在 50°C 和 60°C 下具有更好的热稳定性，比野生型的比活提高了 99.1%，达到 206.61 U/mg。通过分析预测结构，突变体 MuBAA 的耐热性提高主要与通过更多的钙结合位点和围绕 Asp187 的分子内相互作用增强 B 结构域中环区域的稳定性有关。此外，A 结构域中 28 和 370 位点周围的额外分子内相互作用也有利于提高耐热性。此外，活性腔的空间位阻减小增加了突变体 MuBAA 的比活。提高 BAA 的耐热性对 α-淀粉酶的修饰具有理论参考价值。