Focused directed evolution of ß-glucosidases: theoretical versus real effectiveness of a minimal working setup and simple robust screening.
Carbohydr Res
; 346(2): 238-42, 2011 Feb 01.
Article
em En
| MEDLINE
| ID: mdl-21146813
ABSTRACT
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 ß-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 ß-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 oversampling, of the mutant library, which would be necessary to compensate for the underrepresentation of some sequences. This evaluation of the overall performance of the method allows experimentally derived parameters to be incorporated into mutant library design. This method combines the application of a well-defined distribution of variability with a reliable screening process. Thus, it facilitates the production of novel functional variants of ß-glucosidases for either fundamental studies or potential biotechnological applications.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Evolução Molecular Direcionada
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Celulases
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Aminoácidos
Tipo de estudo:
Diagnostic_studies
/
Screening_studies
Idioma:
En
Ano de publicação:
2011
Tipo de documento:
Article