定点突变提高枯草芽孢杆菌角蛋白酶的低温催化活性

Improving the low-temperature activity of Bacillus subtilis keratinase by site-directed mutagenesis

摘要:

[背景] 角蛋白酶KerZ1能在60 ℃的最适温度下高效降解角蛋白底物，然而其在低于最适温度条件下的酶活极低，难以适应工业生产和实际应用的要求。[目的] 提升角蛋白酶KerZ1的低温催化活性。[方法] 结合同源比对与折叠自由能分析向角蛋白酶KerZ1引入氨基酸突变，并对突变体的酶学性质进行研究。[结果] 对KerZ1柔性环区域（loop 13）引入的氨基酸突变T210S、N211S、T212G均使其低温催化活性提升。随后对loop 13进行了复合突变并构建了三突变体T210S/N211S/T212G，该突变体在中等温度（40 ℃）和低温（20 ℃）条件下的比酶活较KerZ1分别提升了45.68%和85.74%。同时，该突变体在60 ℃的半衰期（t_1/2）仅下降11.52%，说明突变体T210S/N211S/T212G在不严重损失热稳定性的情况下其低温催化活性得到了显著的提高。[结论] 氢键的减少及氨基酸疏水性降低导致的蛋白质分子柔性的提升，可能是导致突变体酶T210S/N211S/T212G低温催化活性提升的主要原因。本研究从实际应用出发对角蛋白酶进行低温催化活性改造，为其工业化与应用化奠定了基础。

关键词:角蛋白酶;低温催化活性;分子改造;同源比对;分子动力学模拟;分子柔性;氢键

Abstract:

[Background] The keratinase KerZ1 could efficiently degrade keratin at the optimum temperature of 60℃. However, the poor activity below the optimum temperature makes the enzyme difficult to be applied in industrial production. [Objective] To improve the low-temperature activity of KerZ1. [Methods] Based on homologous alignment and analysis of folding free energy, site-directed mutagenesis was employed for KerZ1, and then the enzymatic properties of the mutants were characterized. [Results] The introduction of T210S, N211S, or T212G into the flexible loop region (loop 13) increased the low-temperature activity of KerZ1. Subsequently, the compound mutant T210S/N211S/T212G was constructed, which showed the activity 45.68% and 85.74% higher than that of KerZ1 at moderate (40℃) and low (20℃) temperatures, respectively. Meanwhile, the half-life (t_1/2) at 60℃ decreased by only 11.52%, indicating that the low-temperature activity of T210S/N211S/T212G was improved without serious loss of thermostability. [Conclusion] The reduction of hydrogen bonds and surface hydrophobicity may be the main reasons for the increased flexibility of the keratinase molecule, which enhances the low-temperature activity of T210S/N211S/T212G. In summary, this study modifies the keratinase to improve its low-temperature activity and lays a foundation for the practical application of this enzyme.

Key words:keratinase;low-temperature activity;molecular modification;homologous alignment;molecular dynamics simulation;molecular flexibility;hydrogen bond