定点突变提高苯丙氨酸羟化酶的热稳定性

Thermal stability improvement for phenylalanine hydroxylase by site-directed mutagenesis

摘要:

嗜热菌中，蛋白质存在Ala替换Gly以及Arg替换Lys的趋势。为了提高紫色色杆菌来源的苯丙氨酸羟化酶的热稳定性，将该酶中所有Gly突变成Ala，Lys突变成Arg，筛选获得热稳定性提高的突变体，并进行组合突变，对突变酶的酶学性质进行研究。结果表明，突变酶K94R和G221A在50 ℃的半衰期分别为26.2 min、16.8 min，比原始酶 (9.0 min) 分别提高了1.9倍、0.9倍，同时组合突变酶K94R/G221A在50 ℃处理1 h后仍保留65.6%的酶活，比原始酶 (8.6%) 高出6.6倍。圆二色谱结果显示原始酶和突变酶K94R、G221A及K94R/G221A的Tm值分别为51.5 ℃、53.8 ℃、53.1 ℃和54.8 ℃。蛋白三维结构模拟推测突变体热稳定性提高机理为：突变体K94R中Arg94与Ile95之间形成额外氢键，稳定其所在的柔性区域；突变体G221A中Ala221与Leu281产生疏水作用，稳定酶分子C-端柔性区。该研究结果为蛋白质热稳定性改造提供了参考，也为苯丙氨酸羟化酶在功能性食品领域的应用奠定了基础。

Abstract:

In proteins of thermophilic bacteria, Gly is tend to be replaced by Ala and Lys is tend to be replaced by Arg to adapt the high temperature. In order to improve the thermal stability of phenylalanine hydroxylase (PAH) from Chromobacterium violaceum, all the Gly on PAH were mutated to Ala and Lys to Arg. Positive mutant enzymes with improved thermal stability were selected, followed by combined mutation and characterization. The results revealed that half-lives of K94R and G221A mutants at 50 °C were 26.2 min and 16.8 min, which were increased by 1.9-times and 0.9-times than the parent enzyme (9.0 min). The residual activity of K94R/G221A mutant was improved to 65.6% after keeping at 50 °C for 1 h, which was 6.6 time higher than the parent enzyme (8.6%). Circular dichroism (CD) spectroscopy revealed that Tm values of the parent enzyme, K94R, G221A and K94R/G221A were 51.5 ℃, 53.8 ℃, 53.1 ℃ and 54.8 ℃, respectively. According to the protein structure simulation, the two mutations were located on flexible loop. In the K94R mutant, the mutated Arg94 on the surface of the enzyme formed an extra hydrogen bond with Ile95, which stabilized the located loop. In the G221A mutant, the mutated Ala221 formed hydrophobic interaction with Leu281, which could stabilize both the loop and flexible area of the C-terminus of G221A. The results not only provided a reference for protein modification on thermal stability, but also laid the foundation for application of phenylalanine hydroxylase in the field of functional foods.