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Development of chimeric laccases by directed evolution

AutorPardo, Isabel ; Vicente, Ana I. ; Maté, Diana M. ; Alcalde Galeote, Miguel ; Camarero, Susana
Palabras claveChimeric laccases
DNA shuffling
α-factor prepro-leader
High-throughput screening
S. cerevisiae
Fecha de publicación12-jul-2012
EditorJohn Wiley & Sons
CitaciónBiotechnol Bioeng. 109(12):2978-86 (2012 )
ResumenDNA 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 α-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 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
Descripción28 p.-4 fig.-1 fig.supl.
Versión del editorhttp://dx.doi.org/ 10.1002/bit.24588
URIhttp://hdl.handle.net/10261/133997
DOI10.1002/bit.24588
ISSN0006-3592
1097-0290
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