Enhancing the Thermostability of Feruloyl Esterase EstF27 by Directed Evolution and the Underlying Structural Basis.
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.
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.
Li-chuang
LC
Cao
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.
Ran
R
Chen
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.
Wei
W
Xie
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.
Yu-huan
YH
Liu
D000975:Antioxidants; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003373:Coumaric Acids / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray; D004043:Dietary Fiber / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004795:Enzyme Stability; D006358:Hot Temperature; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D016296:Mutagenesis; D013329:Structure-Activity Relationship
D000975:Antioxidants; D003373:Coumaric Acids; D004043:Dietary Fiber; D004220:Disulfides; C004999:ferulic acid; D002265:Carboxylic Ester Hydrolases; C070196:feruloyl esterase
X-ray crystallography; crystal structure; directed evolution; feruloyl esterase; thermostability; wheat bran