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A selection assay for haloalkane dehalogenase activity based on toxic substrates.

Appl. Microbiol. Biotechnol.. 2015; 
FibingerMichael P C,DavidsTimo,BöttcherDominique,BornscheuerU
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Mutagenesis Services … Jiří Damborský from Masaryk University, Brno, Czech Republic (Dvorak et al 2014) The CIF gene (GenBank ABJ121721) was ordered commercially (GenScript, USA) and subcloned in vector pET28a with NcoI and XhoI Site directed and saturation mutagenesis … Get A Quote

摘要

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 10 cells, based on inactive variants of the haloalkane ... More

关键词

Continuous selection process,Growth assay,Haloalkane dehalogenase,Toxi