Directed Evolution of Subtilisin E into a Highly Active and Guanidinium Chloride- and Sodium Dodecylsulfate-Tolerant Protease

ChemBioChem ◽  
2012 ◽  
Vol 13 (5) ◽  
pp. 691-699 ◽  
Author(s):  
Zhenwei Li ◽  
Danilo Roccatano ◽  
Michael Lorenz ◽  
Ulrich Schwaneberg
Keyword(s):  
Directed Evolution ◽  
Sodium Dodecylsulfate ◽  
Guanidinium Chloride ◽  
Highly Active ◽  
Subtilisin E

Insights on activity and stability of subtilisin E towards guanidinium chloride and sodium dodecylsulfate

2014 ◽  
Vol 169 ◽  
pp. 87-94 ◽  
Author(s):  
Zhenwei Li ◽  
Danilo Roccatano ◽  
Michael Lorenz ◽  
Ronny Martinez ◽  
Ulrich Schwaneberg
Keyword(s):  
Sodium Dodecylsulfate ◽  
Guanidinium Chloride ◽  
Subtilisin E

scholarly journals An in vivo selection system with tightly regulated gene expression enables directed evolution of highly efficient enzymes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Parinthon Nearmnala ◽  
Manutsawee Thanaburakorn ◽  
Watanalai Panbangred ◽  
Pimchai Chaiyen ◽  
Narupat Hongdilokkul
Keyword(s):  
Directed Evolution ◽  
Selection Pressure ◽  
Catalytic Efficiency ◽  
Selection System ◽  
In Vivo Selection ◽  
Highly Efficient ◽  
Highly Active ◽  
Wide Range ◽  
Regulated Gene Expression

AbstractIn vivo selection systems are powerful tools for directed evolution of enzymes. The selection pressure of the systems can be tuned by regulating the expression levels of the catalysts. In this work, we engineered a selection system for laboratory evolution of highly active enzymes by incorporating a translationally suppressing cis repressor as well as an inducible promoter to impart stringent and tunable selection pressure. We demonstrated the utility of our selection system by performing directed evolution experiments using TEM β-lactamase as the model enzyme. Five evolutionary rounds afforded a highly active variant exhibiting 440-fold improvement in catalytic efficiency. We also showed that, without the cis repressor, the selection system cannot provide sufficient selection pressure required for evolving highly efficient TEM β-lactamase. The selection system should be applicable for the exploration of catalytic perfection of a wide range of enzymes.

scholarly journals Full-Spectrum Targeted Mutagenesis in Plant and Animal Cells

2021 ◽  
Vol 22 (2) ◽  
pp. 857
Author(s):  
Brian Iaffaldano ◽  
Jakob Reiser
Keyword(s):  
Directed Evolution ◽  
Targeted Mutagenesis ◽  
Structural Proteins ◽  
Biological Research ◽  
Animal Cells ◽  
Full Spectrum ◽  
Functional Studies ◽  
Highly Active ◽  
Powerful Approach

Directed evolution is a powerful approach for protein engineering and functional studies. However, directed evolution outputs from bacterial and yeast systems do not always translate to higher organisms. In situ directed evolution in plant and animal cells has previously been limited by an inability to introduce targeted DNA sequence diversity. New hypermutation tools have emerged that can generate targeted mutations in plant and animal cells, by recruiting mutagenic proteins to defined DNA loci. Progress in this field, such as the development of CRISPR-derived hypermutators, now allows for all DNA nucleotides within user-defined regions to be altered through the recruitment of error-prone DNA polymerases or highly active DNA deaminases. The further engineering of these mutagenesis systems will potentially allow for all transition and transversion substitutions to be generated within user-defined genomic windows. Such targeted full-spectrum mutagenesis tools would provide a powerful platform for evolving antibodies, enzymes, structural proteins and RNAs with specific desired properties in relevant cellular contexts. These tools are expected to benefit many aspects of biological research and, ultimately, clinical applications.

Enhancing the Thermostability of Highly Active and Glucose-Tolerant β-Glucosidase Ks5A7 by Directed Evolution for Good Performance of Three Properties

2018 ◽  
Vol 66 (50) ◽  
pp. 13228-13235 ◽  
Author(s):  
Lichuang Cao ◽  
Shuifeng Li ◽  
Xin Huang ◽  
Zongmin Qin ◽  
Wei Kong ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Highly Active ◽  
Glucose Tolerant

scholarly journals In vitro ribosome synthesis and evolution through ribosome display

2019 ◽  
Author(s):  
Michael J. Hammerling ◽  
Brian R. Fritz ◽  
Danielle J. Yoesep ◽  
Do Soon Kim ◽  
Erik D. Carlson ◽  
...  
Keyword(s):  
Cell Viability ◽  
Directed Evolution ◽  
Ribosome Display ◽  
Highly Active ◽  
Integrated Strategy ◽  
A Cell ◽  
Rapid Generation ◽  
Free Environment ◽  
Selection Of

ABSTRACTDirected evolution of the ribosome for expanded substrate incorporation and novel functions is challenging because the requirement of cell viability limits the mutations that can be made. However, our recent development of an integrated strategy for the in vitro synthesis and assembly of translationally competent ribosomes (iSAT) enables the rapid generation of large libraries of ribosome variants in a cell-free environment. Here we combine the iSAT system with ribosome display to develop a fully in vitro methodology for ribosome synthesis and evolution (called RISE). We validate this method by selecting highly active genotypes which are resistant to the antibiotic clindamycin from a library of ribosome variants. We further demonstrate the prevalence of positive epistasis in successful genotypes, highlighting the importance of such interactions in selecting for new function. We anticipate that RISE will facilitate understanding of molecular translation and enable selection of ribosomes with altered properties.

How directed evolution reshapes the energy landscape in an enzyme to boost catalysis

Science ◽  
2020 ◽  
pp. eabd3623
Author(s):  
Renee Otten ◽  
Ricardo A. P. Pádua ◽  
H. Adrian Bunzel ◽  
Vy Nguyen ◽  
Warintra Pitsawong ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Conformational Changes ◽  
Proton Transfer Reaction ◽  
High Energy ◽  
Conformational Ensemble ◽  
Chemical Transformations ◽  
Catalytic Function ◽  
Highly Active ◽  
Transition State Stabilization ◽  
Speed Up

The advent of biocatalysts designed computationally and optimized by laboratory evolution provides an opportunity to explore molecular strategies for augmenting catalytic function. Applying a suite of NMR, crystallographic, and stopped-flow techniques to an enzyme designed for an elementary proton transfer reaction, we show how directed evolution gradually altered the conformational ensemble of the protein scaffold to populate a narrow, highly active conformational ensemble and achieve a nearly billionfold rate acceleration. Mutations acquired during optimization enabled global conformational changes, including high-energy backbone rearrangements, that cooperatively organized the catalytic base and oxyanion stabilizer, thus perfecting transition-state stabilization. Explicit sampling of conformational sub-states during design, and specifically stabilizing productive over all unproductive conformations, could speed up the development of protein catalysts for many chemical transformations.

Directed evolution of a highly active Yersinia mollaretii phytase

2011 ◽  
Vol 95 (2) ◽  
pp. 405-418 ◽  
Author(s):  
Amol V. Shivange ◽  
Annegret Serwe ◽  
Alexander Dennig ◽  
Danilo Roccatano ◽  
Stefan Haefner ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Highly Active
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