Enhancing the Thermostability of Feruloyl Esterase EstF27 by Directed Evolution and the Underlying Structural Basis
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- 作者：Li-chuang Cao ; Ran Chen ; Wei Xie ; Yu-huan Liu
- 刊名：Journal of Agricultural and Food Chemistry
- 出版年：2015
- 出版时间：September 23, 2015
- 年：2015
- 卷：63
- 期：37
- 页码：8225-8233
- 全文大小：411K
- ISSN：1520-5118
文摘
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 掳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 卤 6.8 and 464.8 卤 11.9 渭M, respectively. To further understand the structural basis of the enhanced thermostability, the crystal structure of M6 is determined at 2.0 脜. 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鈥揻unction relationship of feruloyl esterase.