Enhancing the Thermostability of Feruloyl Esterase EstF27 by Directed Evolution and the Underlying Structural Basis

Journal of Agricultural and Food Chemistry
Li-Chuang CaoYu Huan Liu

Abstract

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.

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Citations

Sep 29, 2019·Analytical Biochemistry·Moemi TatsumiToshimi Mizukoshi
May 13, 2021·Journal of Agricultural and Food Chemistry·Xue WeiFengjiao Xin
Feb 2, 2019·Bioresource Technology·Dyoni M OliveiraWanderley D Dos Santos

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