Cao Li-chuang, Chen Ran, Xie Wei, Liu Yu-huan
School of Life Sciences, ‡State Key Laboratory for Biocontrol, School of Life Sciences, and §South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Sun Yat-sen University , Guangzhou 510275, People's Republic of China.
J Agric Food Chem. 2015 Sep 23;63(37):8225-33. doi: 10.1021/acs.jafc.5b03424. Epub 2015 Sep 10.
To improve the thermostability of EstF27, two rounds of random mutagenesis were performed. A thermostable mutant, M6, with six amino acid substitutions was obtained. The half-life of M6 at 55 °C is 1680 h, while that of EstF27 is 0.5 h. The Kcat/Km value of M6 is 1.9-fold higher than that of EstF27. The concentrations of ferulic acid released from destarched wheat bran by EstF27 and M6 at their respective optimal temperatures were 223.2 ± 6.8 and 464.8 ± 11.9 μM, respectively. To further understand the structural basis of the enhanced thermostability, the crystal structure of M6 is determined at 2.0 Å. Structural analysis shows that a new disulfide bond and hydrophobic interactions formed by the mutations may play an important role in stabilizing the protein. This study not only provides us with a robust catalyst, but also enriches our knowledge about the structure-function relationship of feruloyl esterase.
为提高EstF27的热稳定性，进行了两轮随机诱变。获得了一个具有六个氨基酸取代的热稳定突变体M6。M6在55℃下的半衰期为1680小时，而EstF27的半衰期为0.5小时。M6的催化常数与米氏常数之比（Kcat/Km）比EstF27高1.9倍。在各自的最佳温度下，EstF27和M6从脱淀粉麦麸中释放的阿魏酸浓度分别为223.2±6.8μM和464.8±11.9μM。为了进一步了解热稳定性增强的结构基础，测定了M6在2.0 Å分辨率下的晶体结构。结构分析表明，突变形成的一个新的二硫键和疏水相互作用可能在稳定蛋白质方面发挥重要作用。本研究不仅为我们提供了一种高效的催化剂，还丰富了我们对阿魏酸酯酶结构-功能关系的认识。
