Directed Evolution of Enzyme Robustness

doi:10.1002/9783527655465.ch6

Cited by 3 publications

(1 citation statement)

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“…The identification and insertion of such consensus mutations in a protein template is known as consensus design, and it is thought to help stabilize enzymes while bypassing the unwanted trade-off between activity and stability (Porebski and Buckle, 2016;Siddiqui, 2017). Compared with other strategies to improve thermostability [e.g., directed evolution, rational rigidification of flexible loops, computational methods like FRESCO, SCHEMA or PROSS, ancestral libraries or protein resurrection (Cole and Gaucher, 2011;Nestl and Hauer, 2014;Magliery, 2015;Reetz, 2017)], consensus design has some appealing advantages that make it a good choice to be applied individually or in combination with the aforementioned approaches. Indeed, consensus mutagenesis establishes an optimum balance between library size and the required knowledge of the protein, which allows smart and functionally enriched mutant libraries to be generated from MSAs, while streamlining resources and research efforts (Lehmann et al, 2000(Lehmann et al, , 2002Jackel et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

Consensus Design of an Evolved High-Redox Potential Laccase

Gómez-Fernández

Risso

Sanchez-Ruiz

et al. 2020

Front. Bioeng. Biotechnol.

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Among the broad repertory of protein engineering methods that set out to improve stability, consensus design has proved to be a powerful strategy to stabilize enzymes without compromising their catalytic activity. Here, we have applied an in-house consensus method to stabilize a laboratory evolved high-redox potential laccase. Multiple sequence alignments were carried out and computationally refined by applying relative entropy and mutual information thresholds. Through this approach, an ensemble of 20 consensus mutations were identified, 18 of which were consensus/ancestral mutations. The set of consensus variants was produced in Saccharomyces cerevisiae and analyzed individually, while site directed recombination of the best mutations did not produce positive epistasis. The best single variant carried the consensus-ancestral A240G mutation in the neighborhood of the T2/T3 copper cluster, which dramatically improved thermostability, kinetic parameters and secretion.

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Section: Introductionmentioning

confidence: 99%

Consensus Design of an Evolved High-Redox Potential Laccase

Gómez-Fernández

Risso

Sanchez-Ruiz

et al. 2020

Front. Bioeng. Biotechnol.

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Utility of B-Factors in Protein Science: Interpreting Rigidity, Flexibility, and Internal Motion and Engineering Thermostability

et al. 2019

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The term B-factor, sometimes called the Debye–Waller factor, temperature factor, or atomic displacement parameter, is used in protein crystallography to describe the attenuation of X-ray or neutron scattering caused by thermal motion. This review begins with analyses of early protein studies which suggested that B-factors, available from the Protein Data Bank, can be used to identify the flexibility of atoms, side chains, or even whole regions. This requires a technique for obtaining normalized B-factors. Since then the exploitation of B-factors has been extensively elaborated and applied in a variety of studies with quite different goals, all having in common the identification and interpretation of rigidity, flexibility, and/or internal motion which are crucial in enzymes and in proteins in general. Importantly, this review includes a discussion of limitations and possible pitfalls when using B-factors. A second research area, which likewise exploits B-factors, is also reviewed, namely, the development of the so-called B-FIT-directed evolution method for increasing the thermostability of enzymes as catalysts in organic chemistry and biotechnology. In both research areas, a maximum of structural and mechanistic insights is gained when B-factor analyses are combined with other experimental and computational techniques.

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Stereoselectivity-Tailored, Metal-Free Hydrolytic Dynamic Kinetic Resolution of Morita–Baylis–Hillman Acetates Using an Engineered Lipase–Organic Base Cocatalyst

Xia

Xiang

et al. 2017

ACS Catal.

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Metal-free enantiocomplementary hydrolytic dynamic kinetic resolution of Morita−Baylis−Hillman (MBH) acetates was developed using triethylamine (TEA) as a racemization catalyst and wild-type or engineered lipase B from Candida antarctica (CALB) as stereoselectivity-determining catalyst, leading to chiral MBH alcohols with tailor-made R or S configurations on an optional basis. In the TEA-WT CALB catalysis system, WT CALB displays excellent S enantioselectivity for a series of MBH acetates tested (up to 96% ee and 98% conversion). Reversal of enantioselectivity in favor of (R)-MBH alcohols (95% ee; 95% conversion) was achieved by generating a focused site-specific mutagenesis library composed of less than 20 variants. Molecular modeling explains the origin of stereoselectivity.

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Directed Evolution of Enzyme Robustness

Cited by 3 publications

References 147 publications

Consensus Design of an Evolved High-Redox Potential Laccase

Consensus Design of an Evolved High-Redox Potential Laccase

Utility of B-Factors in Protein Science: Interpreting Rigidity, Flexibility, and Internal Motion and Engineering Thermostability

Stereoselectivity-Tailored, Metal-Free Hydrolytic Dynamic Kinetic Resolution of Morita–Baylis–Hillman Acetates Using an Engineered Lipase–Organic Base Cocatalyst

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