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Title

Evolving thermostability in mutant libraries of ligninolytic oxidoreductases expressed in yeast

AuthorsGarcía-Ruiz, Eva CSIC ORCID ; Maté, Diana M.; Ballesteros Olmo, Antonio CSIC ORCID; Martínez, Ángel T. CSIC ORCID ; Alcalde Galeote, Miguel CSIC ORCID
Issue Date18-Mar-2010
PublisherBioMed Central
CitationMicrobial Cell Factories 9:17 (2010)
Abstract[Background] In the picture of a laboratory evolution experiment, to improve the thermostability whilst maintaining the activity requires of suitable procedures to generate diversity in combination with robust high-throughput protocols. The current work describes how to achieve this goal by engineering ligninolytic oxidoreductases (a high-redox potential laccase -HRPL- and a versatile peroxidase, -VP-) functionally expressed in Saccharomyces cerevisiae.
[Results] Taking advantage of the eukaryotic machinery, complex mutant libraries were constructed by different in vivo recombination approaches and explored for improved stabilities and activities. A reliable high-throughput assay based on the analysis of T50 was employed for discovering thermostable oxidases from mutant libraries in yeast. Both VP and HRPL libraries contained variants with shifts in the T50 values. Stabilizing mutations were found at the surface of the protein establishing new interactions with the surrounding residues.
[Conclusions] The existing tradeoff between activity and stability determined from many point mutations discovered by directed evolution and other protein engineering means can be circumvented combining different tools of in vitro evolution.
Description44 pages, 8 figures, 1 table.-- Provisional PDF.
Publisher version (URL)http://dx.doi.org/10.1186/1475-2859-9-17
URIhttp://hdl.handle.net/10261/22551
DOI10.1186/1475-2859-9-17
ISSN1475-2859
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