Development of chimeric laccases by directed evolution
Development of chimeric laccases by directed evolution
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DOI：
10.1002/bit.24588
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发表时间：
2012-12-01
影响因子：
3.8
通讯作者：
Camarero, Susana
中科院分区：
文献类型：
--
作者：
Pardo, Isabel;Isabel Vicente, Ana;Camarero, Susana
DNA重组方法是定向进化蛋白质工程研究中产生多样性的有用工具。我们已经设计了一个阵列的嵌合漆酶与高氧化还原电位在体外和体内的DNA重组的两种真菌漆酶（从Pycnoporus cinnabarinus和PM 1担子菌），这是以前定制的实验室进化的功能表达在酿酒酵母。漆酶融合基因（包括进化的α-因子prepro-leaders在酵母中分泌）进行了一轮的家庭改组，构建嵌合文库和最好的漆酶杂交体中确定的双重高通量筛选（HTS）测定。使用这种方法，我们确定了嵌合体与多达6个交叉事件在整个序列中，我们得到了积极的混合漆酶的pH活性和热稳定性方面的组合特性。Biotechnol. Bioeng. 2012; 109：29782986. (C)2012 Wiley Periodicals，Inc.
DNA recombination methods are useful tools to generate diversity in directed evolution protein engineering studies. We have designed an array of chimeric laccases with high-redox potential by in vitro and in vivo DNA recombination of two fungal laccases (from Pycnoporus cinnabarinus and PM1 basidiomycete), which were previously tailored by laboratory evolution for functional expression in Saccharomyces cerevisiae. The laccase fusion genes (including the evolved a-factor prepro-leaders for secretion in yeast) were subjected to a round of family shuffling to construct chimeric libraries and the best laccase hybrids were identified in dual high-throughput screening (HTS) assays. Using this approach, we identified chimeras with up to six crossover events in the whole sequence, and we obtained active hybrid laccases with combined characteristics in terms of pH activity and thermostability. Biotechnol. Bioeng. 2012; 109: 29782986. (C) 2012 Wiley Periodicals, Inc.