Directed evolution of enzyme stability

Eijsink, Vincent G. H.; Gåseidnes, Sigrid; Borchert, Torben V.; Burg, Bertus van den

doi:10.1016/j.bioeng.2004.12.003

Cited by 383 publications

(265 citation statements)

References 108 publications

Supporting

Mentioning

249

Contrasting

Unclassified

Order By: Relevance

“…9,38 However, the irreversible aggregation and precipitation propensity of the thermally unfolded states has not been commonly addressed by directed evolution so far. In our study, this phenomenon is the major reason for activity retention of the evolved mutants.…”

Section: Discussionmentioning

confidence: 99%

“…[1][2][3] For instance, intermolecular interactions can lead to significant aggregation and subsequent precipitation, a phenomenon which is difficult to predict. Directed evolution [4][5][6][7][8][9] offers an alternative method for improving the robustness of proteins in biotechnology, 10 with error-prone polymerase chain reaction (epPCR) and/or DNA shuffling being the most popular gene mutagenesis techniques in this endeavor. In many cases this approach is superior to rational design, although at the expense of having to screen large mutant libraries, which is still considered to be the bottleneck of directed evolution in general.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: Factors contributing to increased activity retention

Augustyniak

Brzezinska

Pijning³

et al. 2012

Protein Science

View full text Add to dashboard Cite

Previously, Lipase A from Bacillus subtilis was subjected to in vitro directed evolution using iterative saturation mutagenesis, with randomization sites chosen on the basis of the highest B-factors available from the crystal structure of the wild-type (WT) enzyme. This provided mutants that, unlike WT enzyme, retained a large part of their activity after heating above 65°C and cooling down. Here, we subjected the three best mutants along with the WT enzyme to biophysical and biochemical characterization. Combining thermal inactivation profiles, circular dichroism, X-ray structure analyses and NMR experiments revealed that mutations of surface amino acid residues counteract the tendency of Lipase A to undergo precipitation under thermal stress. Reduced precipitation of the unfolding intermediates rather than increased conformational stability of the evolved mutants seems to be responsible for the activity retention.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: Factors contributing to increased activity retention

Augustyniak

Brzezinska

Pijning³

et al. 2012

Protein Science

View full text Add to dashboard Cite

show abstract

“…Although various individual structural features have been identified from those studies, they are used unpredictably to different extents in different proteins. Another strategy to improve protein thermal stability is directed evolution (8,9). Despite many successes and recent technology development, directed evolution still has some practical limitations and thus can be labor-intensive and expensive.…”

mentioning

confidence: 99%

Bioinformatic method for protein thermal stabilization by structural entropy optimization

Bae¹,

Bannen²,

Phillips

2008

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Engineering proteins for higher thermal stability is an important and difficult challenge. We describe a bioinformatic method incorporating sequence alignments to redesign proteins to be more stable through optimization of local structural entropy. Using this method, improved configurational entropy (ICE), we were able to design more stable variants of a mesophilic adenylate kinase with only the sequence information of one psychrophilic homologue. The redesigned proteins display considerable increases in their thermal stabilities while still retaining catalytic activity. ICE does not require a three-dimensional structure or a large number of homologous sequences, indicating a broad applicability of this method. Our results also highlight the importance of entropy in the stability of protein structures.adenylate kinase ͉ improved configurational entropy ͉ local structural entropy ͉ protein engineering ͉ protein stability

show abstract

“…To check whether substrate acceptance and enantioselectivity were evolved at the expense of the robustness of PAMO, T 60 50 values were measured, the temperature at which 50% of the activity has been lost following a heat treatment for 60 min (33,34). The results show that thermostability is essentially maintained: WT PAMO (T 60 50 ¼ 58.2°C); variant Gln93Asn/Pro94Asp (T 60 50 ¼ 56.2°C).…”

Section: Resultsmentioning

confidence: 99%

Induced allostery in the directed evolution of an enantioselective Baeyer–Villiger monooxygenase

Acevedo

Reetz

2010

Proc. Natl. Acad. Sci. U.S.A.

121

118

View full text Add to dashboard Cite

The molecular basis of allosteric effects, known to be caused by an effector docking to an enzyme at a site distal from the binding pocket, has been studied recently by applying directed evolution. Here, we utilize laboratory evolution in a different way, namely to induce allostery by introducing appropriate distal mutations that cause domain movements with concomitant reshaping of the binding pocket in the absence of an effector. To test this concept, the thermostable Baeyer-Villiger monooxygenase, phenylacetone monooxygenase (PAMO), was chosen as the enzyme to be employed in asymmetric Baeyer-Villiger reactions of substrates that are not accepted by the wild type. By using the known X-ray structure of PAMO, a decision was made regarding an appropriate site at which saturation mutagenesis is most likely to generate mutants capable of inducing allostery without any effector compound being present. After screening only 400 transformants, a double mutant was discovered that catalyzes the asymmetric oxidative kinetic resolution of a set of structurally different 2-substituted cyclohexanone derivatives as well as the desymmetrization of three different 4-substituted cyclohexanones, all with high enantioselectivity. Molecular dynamics (MD) simulations and covariance maps unveiled the origin of increased substrate scope as being due to allostery. Large domain movements occur that expose and reshape the binding pocket. This type of focused library production, aimed at inducing significant allosteric effects, is a viable alternative to traditional approaches to "designed" directed evolution that address the binding site directly.allosteric effects | enzymes | molecular dynamics simulations | protein engineering P rotein allostery has been recognized as a positive or negative cooperative event, leading to a structural change at the binding site as a result of distal docking of a molecule acting as an effector (1-5). Allosteric effects can be influenced by such factors as variation in pH, temperature, ionic strength, and covalent modification as well as mutational changes. A variety of experimental and computational techniques have been utilized to unravel the intricacies of this phenomenon (1-5), but also to achieve useful applications such as the creation of protein switches (6). Among the techniques that have been applied to address these challenges is directed evolution, a method that is generally used to engineer the catalytic profiles of enzymes or the binding properties of proteins (7-11). In the endeavor to make directed evolution of enantioselective enzymes more efficient than in the past, we recently introduced the concept of iterative saturation mutagenesis according to which sites around the binding pocket are randomized iteratively, a given site being composed of one or more amino acid positions in the protein (12-14.) When applying this technique to enzymes displaying strong allosteric effects or coupled motions along the reaction coordinate (15), it is necessary to consider such phenomena to make reasonable...

show abstract

Directed evolution of enzyme stability

Cited by 383 publications

References 108 publications

Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: Factors contributing to increased activity retention

Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: Factors contributing to increased activity retention

Bioinformatic method for protein thermal stabilization by structural entropy optimization

Induced allostery in the directed evolution of an enantioselective Baeyer–Villiger monooxygenase

Contact Info

Product

Resources

About