RESUMEN
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(6) 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.
Asunto(s)
Alquenos/metabolismo , Bioensayo/métodos , Escherichia coli/crecimiento & desarrollo , Pruebas Genéticas/métodos , Hidrocarburos Halogenados/metabolismo , Hidrolasas/análisis , Hidrolasas/genética , Alquenos/toxicidad , Escherichia coli/genética , Hidrocarburos Halogenados/toxicidad , Proteínas Mutantes/análisis , Proteínas Mutantes/genéticaRESUMEN
The green and environmentally friendly synthesis of highly valuable organic substances is one possibility for the utilization of laccases (EC 1.10.3.2). As reactants for the herein described syntheses, different o-substituted arylamines or arylthiols and 2,5-dihydroxybenzoic acid and its derivatives were used. In this way, the formation of phenothiazines, phenoxazines, and phenazines was achieved in aqueous solution mediated by the laccase of Pycnoporus cinnabarinus in the presence of oxygen. Two types of phenothiazines (3-hydroxy- and 3-oxo-phenothiazines) formed in one reaction assay were described for the first time. The cyclization reactions yielded C-N, C-S, or C-O bonds. The syntheses were investigated with regard to the substitution pattern of the reaction partners. Differences in C-S and C-N bond formations without cyclization are discussed.
Asunto(s)
Escherichia coli/enzimología , Esterasas/química , Fenilbutiratos/química , Biocatálisis , Ácidos Carboxílicos/química , Ácidos Carboxílicos/metabolismo , Escherichia coli/citología , Escherichia coli/metabolismo , Esterasas/metabolismo , Citometría de Flujo , Glicerol/química , Glicerol/metabolismo , Cinética , Estructura Molecular , Fenilbutiratos/metabolismo , Propanoles/química , Propanoles/metabolismo , Estereoisomerismo , Especificidad por SustratoRESUMEN
Protein engineering is the most important method to overcome the limitations of natural enzymes as biocatalysts. The past few years have seen a tremendous increase in novel concepts to facilitate the design of mutant libraries for focused directed evolution mostly guided by advanced bioinformatic tools. In addition, advanced high-throughput methods were developed using, for example, FACS analysis or microfluidic systems. These achievements significantly facilitate the tailor-made design of enzymes to make them suitable for industrial applications.