米根霉α-淀粉酶热稳定性的理性设计

Improving the thermostability of α-amylase from Rhizopus oryzae by rational design

摘要:

真菌α-淀粉酶被广泛应用于麦芽糖浆生产工业，但其热稳定性普遍较差，在制糖工艺中增加了由于酶活力损失而引起的追加生产成本。在充分研究了热稳定性对于真菌α-淀粉酶应用于工业生产的重要性的基础上，为提高米根霉α-淀粉酶 (ROAmy) 的热稳定性，基于酶蛋白B-factor分析和分子动力学模拟，利用重叠PCR技术分别对ROAmy中的3个氨基酸残基G128、K269和G393进行了单点突变及组合突变。结果表明，所获得的7个突变体均比原酶具有更好的热稳定性，其中效果最好的为组合突变体G128L/K269L/G393P，其在55 ℃下的热失活半衰期 (t1/2) 约为原酶的5.63倍。同时，该突变体的最适温度由50 ℃提高到了65 ℃，最大反应速率 (Vmax) 和催化效率 (kcat/Km) 分别提高了65.38%和99.86%。通过蛋白结构功能比较分析，发现氢键数目的增多或脯氨酸在特殊位置中的引入可能是突变体热稳定性得到提高的主要因素。

Abstract:

Fungal α-amylases are widely used in the production of maltose syrup, while additional production costs may be required in the syrup production process due to the loss of enzyme activity, because of the poor thermostability exhibited in this type of enzyme. After deeply studying the importance of thermostability of fungal α-amylases applied in industrial production, with attempt to improve the thermostability of Rhizopus oryzae α-amylase (ROAmy), single-point mutations and combined mutations that based on analysis of B-factor values and molecular dynamics simulations were carried out for amino acid residues G128, K269 and G393 of ROAmy by overlapping PCR. The results showed that all the 7 mutants obtained presented better thermostability than the wild-type enzyme, and the best mutant was G128L/K269L/G393P which showed a 5.63-fold increase in half-life at 55 ℃ compared with the wild-type enzyme. Meanwhile, its optimum temperature increased from 50 ℃ to 65 ℃, the maximum reaction rate (Vmax) and catalytic efficiency (kcat/Km) increased by 65.38% and 99.86%. By comparing and analyzing the protein structure and function between the mutants and the wild-type enzyme, it was found that the increase of the number of hydrogen bonds or the introduction of proline in special position may be the main reasons for the improved thermostability that found in the mutants.