Laboratory Evolution of High-Redox Potential Laccases
Laboratory Evolution of High-Redox Potential Laccases
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DOI：
10.1016/j.chembiol.2010.07.010
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发表时间：
2010-09-24
影响因子：
--
通讯作者：
Alcalde, Miguel
中科院分区：
文献类型：
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作者：
Mate, Diana;Garcia-Burgos, Carlos;Alcalde, Miguel
具有高氧化还原电位的热稳定性漆酶是通过将定向进化与理性方法相结合的策略来设计的。原始漆酶信号序列被a因子前原前导序列取代，相应的融合基因被针对联合实验室进化，旨在改善酿酒酵母的动力学和分泌，同时保留高热稳定性。经过八轮分子进化，漆酶总活性增强了34,000倍，最终产生了OB-1突变体，作为进化过程的最后一个变体，它是一种在温度、pH范围和有机共溶剂方面具有高活性和稳定性的酶。进化的α因子前前导序列的疏水核心突变增强了功能表达，而成熟蛋白中的一些突变通过改变与周围残基的相互作用来提高其催化能力。
Thermostable laccases with a high-redox potential have been engineered through a strategy that combines directed evolution with rational approaches. The original laccase signal sequence was replaced by the a-factor prepro-leader, and the corresponding fusion gene was targeted for joint laboratory evolution with the aim of improving kinetics and secretion by Saccharomyces cerevisiae, while retaining high thermostability. After eight rounds of molecular evolution, the total laccase activity was enhanced 34,000-fold culminating in the OB-1 mutant as the last variant of the evolution process, a highly active and stable enzyme in terms of temperature, pH range, and organic cosolvents. Mutations in the hydrophobic core of the evolved a-factor prepro-leader enhanced functional expression, whereas some mutations in the mature protein improved its catalytic capacities by altering the interactions with the surrounding residues.