Improving kinetic or thermodynamic stability of an azoreductase by directed evolution
| Autor: | Eduardo P. Melo, Vania Sofia Brissos, Lígia O. Martins, Nádia Gonçalves |
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| Jazyk: | angličtina |
| Rok vydání: | 2015 |
| Předmět: |
Models
Molecular Protein Folding Protein Conformation lcsh:Medicine Biochemistry Protein structure Enzyme Stability NADH NADPH Oxidoreductases Amino Acids lcsh:Science 0303 health sciences Multidisciplinary Protein Stability 030302 biochemistry & molecular biology Temperature Directed evolution Quinone Detoxification Pseudomonas putida Stabilization Enzymes Wastewaters Biodegradation Mutant Libraries Thermodynamics Molecular-Cloning Bacillus-Subtilis Research Article Biotechnology Half-Life Directed Evolution Biophysics Biology Forms of Evolution 03 medical and health sciences Environmental Biotechnology Escherichia coli 030304 developmental biology DNA Primers Enzyme Kinetics Evolutionary Biology Point mutation Mutagenesis lcsh:R Laccase Wild type Rational design Proteins Nitroreductases biology.organism_classification High-Throughput Screening Assays Kinetics Enzyme Structure Biocatalysis Mutagenesis Site-Directed Chemical stability lcsh:Q Directed Molecular Evolution Thermostability |
| Zdroj: | Repositório Científico de Acesso Aberto de Portugal Repositório Científico de Acesso Aberto de Portugal (RCAAP) instacron:RCAAP PLoS ONE PLoS ONE, Vol 9, Iss 1, p e87209 (2014) |
| Popis: | Protein stability arises from a combination of factors which are often difficult to rationalise. Therefore its improvement is better addressed through directed evolution than by rational design approaches. In this study, five rounds of mutagenesis/recombination followed by high-throughput screening (approximate to 10,000 clones) yielded the hit 1B6 showing a 300-fold higher half life at 50 degrees C than that exhibited by the homodimeric wild type PpAzoR azoreductase from Pseudomonas putida MET94. The characterization using fluorescence, calorimetry and light scattering shows that 1B6 has a folded state slightly less stable than the wild type (with lower melting and optimal temperatures) but in contrast is more resistant to irreversible denaturation. The superior kinetic stability of 1B6 variant was therefore related to an increased resistance of the unfolded monomers to aggregation through the introduction of mutations that disturbed hydrophobic patches and increased the surface net charge of the protein. Variants 2A1 and 2A1-Y179H with increased thermodynamic stability (10 to 20 degrees C higher melting temperature than wild type) were also examined showing the distinctive nature of mutations that lead to improved structural robustness: these occur in residues that are mostly involved in strengthening the solvent-exposed loops or the inter-dimer interactions of the folded state. European Union [BIORENEW,, FP6-2004-NMP-NI-4/026456]; Fundacao para a Ciencia e Tecnologia, Portugal [PEst-OE/EQB/LA0004/2011, PTDC/QUI-BIQ/119677/2010]; FCT, Portugal [SFRH/BPD/46808/2008] |
| Databáze: | OpenAIRE |
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