期刊
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
卷 63, 期 37, 页码 8225-8233出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.jafc.5b03424
关键词
feruloyl esterase; directed evolution; thermostability; wheat bran; crystal structure; X-ray crystallography
资金
- National Natural Science Foundation of China [31100579, 31170117]
- Guangdong Innovative Research Team Program [2011Y038]
- Science & Technology Projects of Guangdong Provincial Oceanic and Fishery Bureau [A201301C04]
- National Marine Research Special Funds for Public Welfare Projects of China [201205020]
- Major Science and Technology Projects of Guangdong Province, China [2011A080403006]
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 degrees 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 mu M, respectively. To further understand the structural basis of the enhanced thermostability, the crystal structure of M6 is determined at 2.0 angstrom. 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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