Enhancing the thermal robustness of an enzyme by directed evolution: least favorable starting points and inferior mutants can map superior evolutionary Pathways

Gumulya, Yosephine and Reetz, Manfred T. (2011) Enhancing the thermal robustness of an enzyme by directed evolution: least favorable starting points and inferior mutants can map superior evolutionary Pathways. ChemBioChem, 12 16: 2502-2510. doi:10.1002/cbic.201100412


Author Gumulya, Yosephine
Reetz, Manfred T.
Title Enhancing the thermal robustness of an enzyme by directed evolution: least favorable starting points and inferior mutants can map superior evolutionary Pathways
Journal name ChemBioChem   Check publisher's open access policy
ISSN 1439-4227
1439-7633
Publication date 2011-11-04
Sub-type Article (original research)
DOI 10.1002/cbic.201100412
Open Access Status Not yet assessed
Volume 12
Issue 16
Start page 2502
End page 2510
Total pages 9
Place of publication Weinheim, Germany
Publisher Wiley
Language eng
Formatted abstract
In a previous directed evolution study, the B-FIT approach to increasing the thermal robustness of proteins was introduced and applied to the lipase from Bacillus subtilis. It is based on the general concept of iterative saturation mutagenesis (ISM), according to which sites in an enzyme are subjected to saturation mutagenesis, the best hit of a given library is then used as a template for randomization at other sites, and the process is continued until the desired catalyst improvement has been achieved. The appropriate choice of the ISM sites is crucial; in the B-FIT method the criterion is residues characterized by highest B factors available from X-ray crystallography data. In the present study, B-FIT was employed in order to increase the thermal robustness of the epoxide hydrolase from Aspergillus niger. Several rounds of ISM resulted in the best variant showing a 21 °C increase in the equation image value, an 80-fold improvement in half-life at 60 °C, and a 44 kcal mol−1 improvement in inactivation energy. Seven other variants were also evolved with moderate yet significant improvements; these were characterized by 10–14 °C increases in equation image, 20–30-fold improvement in half-lives at 60 °C and 15–20 kcal mol−1 elevations in activation energy. Unexpectedly, in the ISM process the best variants were obtained from essentially neutral or even inferior mutant parents, that is, when a given library contains no improved mutants. This constitutes a practical way to escape from what appear to be local minima (“dead ends”) in the fitness landscape—a finding of notable significance in directed evolution.
Keyword Directed
Enzymes
Evolution
Hydrolases
Neutral drift
Quasi-species
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Non-UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: School of Chemistry and Molecular Biosciences
 
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