基于空腔填充效应构建伯克霍尔德菌脂肪酶LipA的热稳定性突变体

Effect of cavity-filling mutations on thermostability of lipase LipA from Burkholderia sp.

摘要:

[目的]中温伯克霍尔德菌胞外脂肪酶LipA在工业领域具有重要的应用价值。利用蛋白质工程技术来提高其热稳定性，对开发脂肪酶LipA酶制剂及提高其应用范围及应用效果，具有重要的意义。[方法]利用生物信息学软件Castp、Voronoia和Cave分析LipA分子中存在的空腔及其组成氨基酸残基；利用FoldX软件构建上述氨基酸残基的突变体电子文库，并基于空腔效应（体积变小）、自由能变化值（降低）和空间结构特点等对前述突变体电子文库进行筛选。从突变体电子文库中选择具有代表性的突变体，通过基因工程技术，引入突变。经诱导表达后，实验验证并筛选出热稳定性的突变体。[结果]构建了一个由58个突变体组成的电子文库；并对其中17个代表性的突变体进行了实验验证；筛选到2个热稳定性有明显提高的突变体LipA-His¹⁵Pro和LipA-Ala²¹⁰Val；其叠加突变体LipA-His¹⁵Pro/Ala²¹⁰Val的T₅₀¹²较野生型LipA提高了8℃，在55℃下的半衰期较野生型脂肪酶LipA提高了23.1倍。[结论]基于空腔填充技术构建热稳定性伯克霍尔德菌胞外脂肪酶LipA突变体，是一种行之有效的策略。

关键词:伯克霍尔德菌;脂肪酶LipA;空腔填充效应;T₅₀¹²;半衰期

Abstract:

[Objective] To improve the thermostability of lipase LipA from Burkholderia sp., a series of cavity-filled lipase LipA mutants were constructed and evaluated. [Methods] Amino acid residuals consisted of cavities in 3D structural model of lipase LipA were predicted using Castp, Voronoia and Cave, and then computational libraries of every amino acid residuals were constructed using software packages FoldX. The following types of mutants were excluded from the computational libraries:(1) volume of the cavity was increased; (2) value of free energy, ΔΔG, was over -0.5 kcal/mol. Total 58 mutants were screened from the computational libraries, and 17 mutants were selected to verify the mutation effect. [Results] Thermostability of lipase mutants (LipA-His¹⁵Pro and LipA-Ala²¹⁰Val) were improved and T₅₀¹² value of lipase LipA-His¹⁵Pro and lipase LipA-Ala²¹⁰Val increased by 4 ℃ and 2 ℃, respectively. Thermostability of the superimposing mutant lipase LipA-His¹⁵Pro/Ala²¹⁰Val was further improved and the half-life (t_1/2) at 55 ℃ increased by 23.1 times. [Conclusion] Cavity-filling mutation was a feasible technique to improve the thermostability of lipase LipA from Burkholderia sp.

Key words:Burkholderia sp.;lipase LipA;cavity-filling mutation;T₅₀¹²;half-life