Iterative Saturation Mutagenesis Accelerates Laboratory Evolution of Enzyme Stereoselectivity: Rigorous Comparison with Traditional Methods

2010 ◽  
Vol 132 (26) ◽  
pp. 9144-9152 ◽  
Author(s):  
Manfred T. Reetz ◽  
Shreenath Prasad ◽  
José D. Carballeira ◽  
Yosephine Gumulya ◽  
Marco Bocola
Keyword(s):  
Saturation Mutagenesis ◽  
Traditional Methods ◽  
Laboratory Evolution ◽  
Iterative Saturation Mutagenesis

Directed evolution of enantioselective enzymes: An unceasing catalyst source for organic chemistry

2010 ◽  
Vol 82 (8) ◽  
pp. 1575-1584 ◽  
Author(s):  
Manfred T. Reetz ◽  
Sheng Wu ◽  
Huabao Zheng ◽  
Shreenath Prasad
Keyword(s):  
Organic Chemistry ◽  
Directed Evolution ◽  
Saturation Mutagenesis ◽  
Powerful Method ◽  
Laboratory Evolution ◽  
Synthetic Organic Chemistry ◽  
Iterative Saturation Mutagenesis

Directed evolution has emerged as a powerful method for engineering essentially any catalytic parameter of enzymes for application in synthetic organic chemistry and biotechnology, including thermostability, substrate scope, and enantioselectivity. Enantioselectivity is especially crucial when applying biocatalysts to synthetic organic chemistry. This contribution focuses on recent methodology developments in laboratory evolution of stereoselective enzymes, hydrolases, and monooxygenases serving as the enzymes. Specifically, iterative saturation mutagenesis (ISM) has been developed as an unusually effective method to evolve enhanced or reversed enantioselectivity, broader substrate scope, and/or higher thermostability of enzymes.

Directed Evolution of an Enantioselective Enoate-Reductase: Testing the Utility of Iterative Saturation Mutagenesis

2009 ◽  
Vol 351 (18) ◽  
pp. 3287-3305 ◽  
Author(s):  
Despina J. Bougioukou ◽  
Sabrina Kille ◽  
Andreas Taglieber ◽  
Manfred T. Reetz
Keyword(s):  
Directed Evolution ◽  
Saturation Mutagenesis ◽  
Enoate Reductase ◽  
Iterative Saturation Mutagenesis

scholarly journals Enhancing the Promiscuous Phosphotriesterase Activity of a Thermostable Lactonase (GkaP) for the Efficient Degradation of Organophosphate Pesticides

2012 ◽  
Vol 78 (18) ◽  
pp. 6647-6655 ◽  
Author(s):  
Yu Zhang ◽  
Jiao An ◽  
Wei Ye ◽  
Guangyu Yang ◽  
Zhi-Gang Qian ◽  
...  
Keyword(s):  
Catalytic Efficiency ◽  
Dynamics Simulation ◽  
Saturation Mutagenesis ◽  
Divergent Evolution ◽  
Laboratory Evolution ◽  
Content Type ◽  
Hydrolytic Activities ◽  
Geobacillus Kaustophilus ◽  
Amidohydrolase Superfamily

ABSTRACTThe phosphotriesterase-like lactonase (PLL) enzymes in the amidohydrolase superfamily hydrolyze various lactones and exhibit latent phosphotriesterase activities. These enzymes serve as attractive templates forin vitroevolution of neurotoxic organophosphates (OPs) with hydrolytic capabilities that can be used as bioremediation tools. Here, a thermostable PLL fromGeobacillus kaustophilusHTA426 (GkaP) was targeted for joint laboratory evolution with the aim of enhancing its catalytic efficiency against OP pesticides. By a combination of site saturation mutagenesis and whole-gene error-prone PCR approaches, several improved variants were isolated. The most active variant, 26A8C, accumulated eight amino acid substitutions and demonstrated a 232-fold improvement over the wild-type enzyme in reactivity (kcat/Km) for the OP pesticideethyl-paraoxon. Concomitantly, this variant showed a 767-fold decrease in lactonase activity with δ-decanolactone, imparting a specificity switch of 1.8 × 105-fold. 26A8C also exhibited high hydrolytic activities (19- to 497-fold) for several OP pesticides, including parathion, diazinon, and chlorpyrifos. Analysis of the mutagenesis sites on the GkaP structure revealed that most mutations are located in loop 8, which determines substrate specificity in the amidohydrolase superfamily. Molecular dynamics simulation shed light on why 26A8C lost its native lactonase activity and improved the promiscuous phosphotriesterase activity. These results permit us to obtain further insights into the divergent evolution of promiscuous enzymes and suggest that laboratory evolution of GkaP may lead to potential biological solutions for the efficient decontamination of neurotoxic OP compounds.

Crystal structure and iterative saturation mutagenesis of ChKRED20 for expanded catalytic scope

2017 ◽  
Vol 101 (23-24) ◽  
pp. 8395-8404 ◽  
Author(s):  
Feng-Jiao Zhao ◽  
Yun Jin ◽  
Zhongchuan Liu ◽  
Chao Guo ◽  
Tong-Biao Li ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Saturation Mutagenesis ◽  
Iterative Saturation Mutagenesis

Directed Evolution by Using Iterative Saturation Mutagenesis Based on Multiresidue Sites

ChemBioChem ◽  
2013 ◽  
Vol 14 (17) ◽  
pp. 2301-2309 ◽  
Author(s):  
Loreto P. Parra ◽  
Rubén Agudo ◽  
Manfred T. Reetz
Keyword(s):  
Directed Evolution ◽  
Saturation Mutagenesis ◽  
Iterative Saturation Mutagenesis
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