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.

中文翻译：

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通过亲水工程，组氨酸标签优化和静态培养提高CotA漆酶的活性。

枯草芽孢杆菌的CotA蛋白具有漆酶活性。重组CotA的溶解度低，阻碍了其应用。在这项研究中，应用组氨酸标签位置优化和亲水工程技术来提高CotA蛋白的产量和活性。结果表明，CotA在C末端带有标签的蛋白质产量（CH6-CotA）是NH6-CotA（在N末端带有His标签）的蛋白质产量的四倍。然后，通过用亲水性氨基酸取代疏水残基来构建23个单一突变体。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）分别是CH6-CotA的两倍和三倍。最后，构建了具有C末端his标签（CH6-TSY）的三重突变体F2071Y / V403T / P455S。