ArticlePloS one2014
Improving kinetic or thermodynamic stability of an azoreductase by directed evolution.
Article in PloS one, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers, 1 of them a synthesis that pooled it.
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Who cites it
14 citing papers in PubMed, 1 synthesis or guideline pooled it, 41 citations in OpenAlex.
- The diversity of PET degrading enzymes: A systematic review of sequence, structure, and function.Protein science : a publication of the Protein Society · 2025Pooled it
- FireProtDB 2.0: large-scale manually curated database of the protein stability data.Nucleic acids research · 2026Article
- Engineering the kinetic stability of a β-trefoil protein by tuning its topological complexity.Frontiers in molecular biosciences · 2023Article
- Characterization of a Novel FeInternational journal of molecular sciences · 2022Article
- Cell-Free Protein Synthesis for the Screening of Novel Azoreductases and Their Preferred Electron Donor.Chembiochem : a European journal of chemical biology · 2022Article
- Thermostability engineering of industrial enzymes through structure modification.Applied microbiology and biotechnology · 2022Review
- A Brief History of Colour, the Environmental Impact of Synthetic Dyes and Removal by Using Laccases.Molecules (Basel, Switzerland) · 2021Review
- Biophysical and structural studies reveal marginal stability of a crucial hydrocarbon biosynthetic enzyme acyl ACP reductase.Scientific reports · 2021Article
- Wasteful Azo Dyes as a Source of Biologically Active Building Blocks.Frontiers in bioengineering and biotechnology · 2021Article
- Facile measurement of protein stability and folding kinetics using a nano differential scanning fluorimeter.Protein science : a publication of the Protein Society · 2019Article
- Complete Draft Genome Sequence ofGenome announcements · 2017Article
- Effect of Mutation and Substrate Binding on the Stability of Cytochrome P450BM3 Variants.Biochemistry · 2016Article
- Gene-specific amplicons from metagenomes as an alternative to directed evolution for enzyme screening: a case study using phenylacetaldehyde reductases.FEBS open bio · 2016Article
- Rational Design of Disulfide Bonds Increases Thermostability of a Mesophilic 1,3-1,4-β-Glucanase from Bacillus terquilensis.PloS one · 2016Article
Corrections and comments
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Authors and funding
4 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
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 (≈10,000 clones) yielded the hit 1B6 showing a 300-fold higher half life at 50°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°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.
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Registered trials
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