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Focused directed evolution of beta-glucosidases: theoretical versus real effectiveness of a minimal working setup and simple robust screening
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SYSNO ASEP 0440786 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Focused directed evolution of beta-glucosidases: theoretical versus real effectiveness of a minimal working setup and simple robust screening Author(s) Mazura, P. (CZ)
Filipi, T. (CZ)
Souček, P. (CZ)
Brzobohatý, Břetislav (BFU-R) RID, ORCIDNumber of authors 4 Source Title Carbohydrate Research. - : Elsevier - ISSN 0008-6215
Roč. 346, č. 2 (2011), s. 238-242Number of pages 5 s. Publication form Print - P Language eng - English Country NL - Netherlands Keywords Directed evolution ; beta-Glucosidase ; Mutagenesis Subject RIV BO - Biophysics Institutional support BFU-R - RVO:68081707 UT WOS 000287111400011 DOI 10.1016/j.carres.2010.08.016 Annotation Here we present an optimized procedure to generate amino acid variations at specific site(s) of proteins, followed by a simple one-step screen for mutants with the desired beta-glucosidase activity. The procedure was evaluated by introducing sequence variation into a codon specifying a non-functional variant of the catalytic nucleophile (E401) of the maize beta-glucosidase Zm-p60.1. Observed and theoretically expected frequencies of the four possible variants of the codon and the two possible phenotypes (functional and non-functional) were investigated. Deviations in codon and phenotype frequencies were expressed as a coefficient. This coefficient was then used to estimate the extent of over-sampling, of the mutant library, which would be necessary to compensate for the underrepresentation of some sequences. Workplace Institute of Biophysics Contact Jana Poláková, polakova@ibp.cz, Tel.: 541 517 244 Year of Publishing 2015
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