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

• Published in: Journal of agricultural and food chemistry Vol. 63; no. 37; pp. 8225 - 8233
• Main Authors: Cao, Li-chuang, Chen, Ran, Xie, Wei, Liu, Yu-huan
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 23.09.2015
• Subjects: Antioxidants; Carboxylic Ester Hydrolases - chemistry; Carboxylic Ester Hydrolases - genetics; Carboxylic Ester Hydrolases - metabolism; Coumaric Acids - metabolism; Crystallization; Crystallography, X-Ray; Dietary Fiber - metabolism; Disulfides - chemistry; Enzyme Stability; Hot Temperature; Hydrophobic and Hydrophilic Interactions; Kinetics; Models, Molecular; Molecular Structure; Mutagenesis; Structure-Activity Relationship; X-ray crystallography; crystal structure; wheat bran; thermostability; feruloyl esterase; directed evolution
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/acs.jafc.5b03424

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 K cat/K m 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.