Gomez-Fernandez Bernardo J, Risso Valeria A, Sanchez-Ruiz Jose M, Alcalde Miguel
Department of Biocatalysis, Institute of Catalysis, CSIC, Madrid, Spain.
Facultad de Ciencias, Departamento de Química Física, Universidad de Granada, Granada, Spain.
Front Bioeng Biotechnol. 2020 May 6;8:354. doi: 10.3389/fbioe.2020.00354. eCollection 2020.
Among the broad repertory of protein engineering methods that set out to improve stability, consensus design has proved to be a powerful strategy to stabilize enzymes without compromising their catalytic activity. Here, we have applied an in-house consensus method to stabilize a laboratory evolved high-redox potential laccase. Multiple sequence alignments were carried out and computationally refined by applying relative entropy and mutual information thresholds. Through this approach, an ensemble of 20 consensus mutations were identified, 18 of which were consensus/ancestral mutations. The set of consensus variants was produced in and analyzed individually, while site directed recombination of the best mutations did not produce positive epistasis. The best single variant carried the consensus-ancestral A240G mutation in the neighborhood of the T2/T3 copper cluster, which dramatically improved thermostability, kinetic parameters and secretion.
在旨在提高稳定性的众多蛋白质工程方法中，共有序列设计已被证明是一种在不影响酶催化活性的情况下稳定酶的有效策略。在此，我们应用一种内部共有序列方法来稳定实验室进化的高氧化还原电位漆酶。通过应用相对熵和互信息阈值进行了多序列比对并进行了计算优化。通过这种方法，鉴定出了20个共有序列突变体，其中18个是共有序列/祖先突变。在[具体宿主]中产生了一组共有序列变体并分别进行了分析，而最佳突变的定点重组并未产生正向上位效应。最佳的单个变体在T2/T3铜簇附近携带共有序列-祖先A240G突变，这显著提高了热稳定性、动力学参数和分泌水平。
