Li Lei, Xie Tian, Liu Zhongchuan, Feng Hong, Wang Ganggang
Key Laboratory of Bio-resources and Eco-environment, Ministry of Education, Sichuan Key Laboratory of Molecular Biology and Biotechnology, College of Life Sciences, Sichuan University, 610064 Chengdu, China.
Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China.
Protein Eng Des Sel. 2018 Jan 1;31(1):1-5. doi: 10.1093/protein/gzx064.
CotA protein from Bacillus subtilis is of laccase activity. The solubility of recombinant CotA is low, which hinders its application. In this study, histidine tag position optimization and hydrophilic engineering were applied to increase the yield and activity of CotA protein. The results showed that the protein yield of CotA with his tag at C-terminal (CH6-CotA) was four times of that of NH6-CotA (His tag at N-terminal). Then, 23 single mutants were constructed by substitutions of hydrophobic residues with hydrophilic amino acids. Among them, the protein yield of the mutant F207Y was increased by 30%; the catalytic activity (kcat/Km) of V403T and P455S was two and three times higher than that of CH6-CotA, respectively. Finally, triple mutant F2071Y/V403T/P455S with C-terminal his-tag (CH6-TSY) was constructed. When the proteins were expressed in microanaerobic condition, the activities of mutants CH6-P455S and CH6-TSY were enhanced about 48- and 42-folds compared to that of NH6-CotA in non-static culture.
来自枯草芽孢杆菌的CotA蛋白具有漆酶活性。重组CotA的溶解度较低，这阻碍了其应用。在本研究中，通过组氨酸标签位置优化和亲水工程来提高CotA蛋白的产量和活性。结果表明，C端带有组氨酸标签的CotA（CH6-CotA）的蛋白产量是N端带有组氨酸标签的NH6-CotA的四倍。然后，通过用亲水性氨基酸取代疏水性残基构建了23个单突变体。其中，突变体F207Y的蛋白产量提高了30%；V403T和P455S的催化活性（kcat/Km）分别比CH6-CotA高两倍和三倍。最后，构建了C端带有组氨酸标签的三突变体F207Y/V403T/P455S（CH6-TSY）。当在微厌氧条件下表达蛋白时，与非静态培养中的NH6-CotA相比，突变体CH6-P455S和CH6-TSY的活性分别提高了约48倍和42倍。
