A selection assay for haloalkane dehalogenase activity based on toxic substrates
A selection assay for haloalkane dehalogenase activity based on toxic substrates
复制标题
基于毒性底物的卤代烷脱卤酶活性选择测定
DOI：
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发表时间：
2015
影响因子：
5
通讯作者：
U. Bornscheuer
中科院分区：
文献类型：
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作者：
Michael P. C. Fibinger;Timo Davids;D. Böttcher;U. Bornscheuer
基于自然选择和进化适应的适者生存，开发了一个智能的高通量系统，从大量突变体库中选择活性卤烷脱卤酶变体。只有活性的酶变体才能水解有毒的卤代烷烃以促进生长，而无活性的突变体则会因有毒化合物而挨饿或死亡。有了这个强大的工具，可以在几天内筛选出巨大的酶突变文库。这种选择不需要任何人工底物，这些底物很难合成，它们也类似于典型的卤代烷烃脱卤酶。基于卤代烷烃去卤酶DhaA或DhlA的失活变体，成功筛选了三个超过106个细胞大小的饱和文库，以检索活性酶。活性野生型酶的富集程度是无活性野生型酶的340倍。此外，这种选择方法可用于连续定向进化实验，以富集表达适应性卤烷脱卤酶的细胞。
Based on natural selection and the survival of the fittest by evolutionary adaption, a smart high-throughput system was developed to select active haloalkane dehalogenase variants from a large mutant library. Only active enzyme variants can hydrolyse toxic halogenated alkanes to promote growth, whereas inactive mutants starve or die due to the toxic compound. With this powerful tool, huge enzyme mutant libraries can be screened within a few days. The selection is done without any artificial substrates that are hard to synthesize and they also resemble typical ones for haloalkane dehalogenases. Three saturation libraries, with a size of more than 106 cells, based on inactive variants of the haloalkane dehalogenases DhaA or DhlA were successfully screened to retrieve active enzymes. The enrichment of the active wild-type enzyme in contrast to the inactive variants was about 340-fold. In addition, this selection approach can be applied for continuous directed evolution experiments for the enrichment of cells expressing adapted haloalkane dehalogenases.
影响因子：
3.3
作者：
D. F. Steele;S. Jinks-Robertson
通讯作者：
D. F. Steele;S. Jinks-Robertson